My Little Pony: Equestria Girls follows Princess Twilight Sparkle in the parallel world, which is accessed through a magic mirror. Together, with the counterparts of her pony friends, Rainbow Dash, Pinkie Pie, Applejack, Rarity and Fluttershy, along with her assistant Spike, Twilight will have to deal with the various magical happenings in Canterlot High.

Later films introduces two additional main characters serving as substitutes for Twilight (due to her duties in Equestria as the Princess of Friendship): Sunset Shimmer, a former student of Princess Celestia; and Twilight "Sci-Twi" Sparkle, the science-inclined parallel world counterpart of Princess Twilight Sparkle.