|Hair||Moderate sapphire blue with moderate violet and Brilliant rose streaks|
|Skin||Pale, light grayish heliotrope|
|Voice||Tara Strong (speaking voice)|
Rebecca Shoichet (singing voice)
Twilight is a pony and human in the other world of Equestria. She has purple eyes, wears a light blue shirt and purple skirt with sparkle like her cutie marks in pony form and purple cold legs and Purple and pink legwarmers with black dress shoes. In the alternate world, she is turned into a teenage human and Spike into a dog. She can't get the hang of being human. Eventually, she meets the human versions of her Ponyville friends and manages to repair the friendships that Sunset Shimmer, who she finds out that everyone in the school in the human world is afraid of due to her meanness, had broken before Twilight arrived. Twilight decides to run against Sunset for Princess of the Fall Formal in order to get her crown back since Fluttershy had given it to the human version of Princess Celestia. She also develops a crush on Sunset's ex-boyfriend Flash Sentry.
Sunset tries many ways to stop Twilight from getting the crown, such as having the human versions of Snips and Snails record Twilight's difficulties coping with human varieties and turning it into a ridiculing video for all of the students to see. This turns Twilight into a laughing stock, but her new friends help turn her popularity around with a song, Equestria Girls. Sunset also has the Fall Formal decorations destroyed and the blame pinned on Twilight, nearly ruining her chances to get the crown back and return home. However, again with the help of Twilight's new best friends, as well as many students in the school, the auditorium is cleaned and redecorated in time for the dance.
At the Fall Formal, Twilight accepts Flash Sentry's offer to dance. She is voted the Fall Formal Princess, but when Snips and Snails capture Spike, she and her best friends chase the duo outside where Sunset Shimmer, in a last ditch effort to get the crown, threatens to destroy the portal leading to Equestria unless Twilight surrenders the crown. Twilight refuses, confident that Equestria can protect itself without the Elements of Harmony, but the evil unicorn-turned-human takes it by force and transforms into a demon, hypnotizing the students and teachers of the school to be her army. Twilight is nearly killed when Sunset Shimmer throws a fireball at her, but the magic crown, powered by her friendship with her gang, protects her and her best friends.
Twilight and her new friends manage to defeat the villainess with the power of friendship, and when Sunset Shimmer is reverted to her human form, she becomes remorseful, stating that she doesn't know where to begin with friendship since she's never had a friend. A forgiving Twilight has her new friends teach its magic to her. Principal Celestia gives Twilight her crown after she and the student body witnessed her true duty of being a princess, no matter which world she was from, can inspire others to stand with her.
After the dance, Twilight and Spike return to Equestria, Twilight feeling more confident about being a princess than before. She bumps into the pony version of Flash Sentry and blushes upon meeting him. Twilight's pony friends tease her for having a crush, but she denies it.
It is implied that Twilight has her own human counterpart in the mirror world; the human Pinkie mentions seeing a girl who looks like her (and owns a dog named Spike) in the city.
The human counterpart of Twilight Sparkle makes a cameo in Rainbow Rocks, appearing in a bonus scene after the credits. She appears to be a scientist who has been researching the strange events going on in her city, with all evidence pointing to Canterlot High School as the source. If further Equestria Girls stories are made, her investigations will likely become a plot point.
When entering the human world, Twilight comes across as a typical socially-awkward and clumsy teenage girl who tries to fit in, which is due to her lack of knowledge of the human world. However, we see that she has her determination when she finds the crown, her studiousness when she leads the students to clean up and redecorate the gym for Fall Formal, and her kind heart when she rebuilds the broken friendships of the human versions of her friends in Ponyville caused by Sunset Shimmer. She also becomes very shy around Flash Sentry due to her crush on him, but manages to work up the confidence to accept his offer to dance with her.
Twilight Sparkle's Quotes
"My magic! It isn't working!"
"What does the rest of me look like?"
Twilight Sparkle: "Looks like if I want my crown back, I'll have to become Princess of the Canterlot High Fall Formal. So that's what I'm gonna do!"
Spike: "And how exactly do you plan on doing that?"
Twilight Sparkle: "'I have no idea!"
"Now would be a good time to start."
"The crown maybe upon your head Sunset Shimmer. But you cannot wield it, because you do not possess the most powerful magic of all....the magic of friendship!"
"The magic of friendship doesn't just exist in Equestria. It's everywhere. You can seek it out, or you can forever be alone."
"I bet they can teach you."
"I know we have only been friends for a short time, but I am going to miss all of you so much."
"I think she's going to be fine. I left her in good hands."
(Twilight's mirror world counterpart, regarding her investigation into CHS) "No doubt about it, Spike. There's definitely something strange going on at that school..."