Twilight Sparkle is the main protagonist of My Little Pony: Friendship is Magic and also one of the seven supporting characters in My Little Pony: The Book of Golden Oak, who made her first debut in season 1 episode 7 "Kunoichi in Peril". She is a pretty unicorn mare who transforms into an Alicorn mare and becomes a princess in Magic Mystery Cure and a pony with a lavender fur coat, purple eyes, and an indigo mane and tail with a pink and purple stripe running through each. She is the younger sister of Shining Armor, the wife of Flash Sentry, the mother of Flashlight, the wife of Flash Sentry, the sister-in-law of Princess Cadance, and the youngest daughter of both Night Light/Mr. Sparkle and Twilight Velvet/Mrs. Sparkle. She also is the paternal aunt of Princess Flurry Heart. She is partnered with Hiro Hamada (Ryan Potter).
She is both a Princess of Friendship and the bearer of the Element of Magic. In the past, she was Celestia's personal student. She lives in her own place in Ponyville, with her baby dragon assistant Spike. In Big Hero 6: Armed Mares, she is serves as Hiro Hamada's partner. In My Little Pony: The Book of Golden Oak, she serves as the acting director of the Golden Oak Library. She has an easy-going nature and a lust for vengeance and battle. She is always searching for an opponent strong enough to defeat her. Before she met Flash Sentry (Vincent Tong), she used to had a relationship with a light brown unicorn stallion named Doughnut Joe (also voiced by Vincent Tong).
- Shining Armor (older brother)
- Princess Cadence (sister-in-law)
- Flashlight (son)
- Flash Sentry (husband)
- Night Light (father)
- Twilight Velvet (mother)
- Flurry Heart (niece)
- Phillip Sentry (father-in-law)
- Mrs. Sentry (mother-in-law)
- Lady Tremaine - Her nemesis in My Little Pony: A Twist in Time. Plans to use her as bait to lure in her eldest daughter Drizella (Russi Taylor).
- Drizella Tremaine - One of her main targets and enemies.
- Anastasia Tremaine - Briefly works alongside her.
- Cinderella - Briefly works alongside her.
- Max Goof - Briefly works alongside him.
- Jafar - One of her main targets and enemies.
- Iago - Briefly works alongside him in My Little Pony: The Revenge of Jafar.
- Goofy Goof - Briefly works alongside him.
- Bradley "Brad" Uppercrust III - One of her main targets and enemies.
- Daisy Duck - Briefly works alongside her.
- Jose Carioca - Briefly works alongside him.
- Panchito Pistoles - Briefly works alongside him.
- Minnie "Minerva" Mouse - Briefly works alongside her.
- Donald Duck - Targets him for his magic staff in Hasbro x Disney.
- Zeke Midas Wolf - One of her main enemies.
- In My Little Pony: Magic Art Online, Twilight leaves a clue saying "Tony Who?" which was a jab at Donald Duck's current English-language voice actor Tony Anselmo.
- Also in My Little Pony: Dragon Layer, Twilight leaves a clue saying "Tress Who?" which was a jab at Daisy Duck's current English-language voice actress Tress MacNeille.