De Villota's sister, Isabel, said an autopsy indicated that she had died in her sleep around 6 a.m. local time due to "neurological damage" from her crash in July 2012. De Villota lost an eye in the accident but had since seemingly recovered from the wreck — even driving again, writing a book about it, and recently getting married.
Spanish police said her death was from "natural causes" and that there was no indication of foul play. They said De Villota's manager alerted staff at the Hotel Sevilla Congresos after her body was found.
De Villota barely survived last year's crash during testing for the Marussia F1 team in England, losing her right eye and sustaining other serious head injuries that kept her hospitalized for a month.
De Villota, a Madrid native, was the daughter of Emilio de Villota, who competed in F1 from 1976-82.
Her family used de Villota's Facebook page to say "Dear friends: Maria has left us. She had to go to heaven like all angels. I give thanks to God for the year and a half that he left her with us."
F1 officials and drivers at the Japanese Grand Prix were stunned by her death.
"My deepest condolences go to the de Villota family," said FIA president Jean Todt. "Maria was a fantastic driver, a leading light for women in motorsport and a tireless campaigner for road safety. Above all she was a friend I deeply admired."
McLaren team principal Martin Whitmarsh said as the chairman of the Formula One Teams' Association the "whole paddock is very shocked by the news that Maria is no longer with us.
"She was an inspiration not just to women in this sport, but also to all those who suffered life-threatening injuries."
Sauber's Monisha Kaltenhorn, the first female team principal in F1, said, "If anybody represented strength and optimism, it was Maria. Her sudden death is a big loss to the motorsport world."
Williams development driver Susie Wolff recalled how de Villota asked her to carry on for her and all women drivers following her accident.
"She very much said to me after it, 'It's up to you to go out there and show them that it (a woman driver in F1) is possible,'" Wolff said. "She knew that women could compete at that level and that's why, after her accident and her not being able to do that anymore, she just wanted someone to know it was possible. She had such a spirit for life. What she came through was a testament to her strength of character and her positive outlook on life."
Marussia expressed its condolences.
"It is with great sadness that we learned a short time ago of the news that Maria de Villota has passed away," Marussia said in a statement. "Our thoughts and prayers are with Maria's family and friends at this very difficult time."
Fellow Spaniard Fernando Alonso said: "It's very sad news for the world of motorsport as Maria was loved by everyone. Now, all we can do is pray for her and for her family."
De Villota also had driven in the world touring car championship in 2006 and 2007 plus the Superleague open-wheel series.
She was in Seville to participate in the conference "What Really Matters," whose mission is to inspire and teach young people "universal human values," in the words of the organizers.
Organizers cancelled the conference on receiving news of her death and issued a statement "transmitting their care and support to the family and loved ones of Maria de Villota."
De Villota was the first Spanish woman to drive an F1 car. Sport minister Jose Wert announced that she would be posthumously awarded Spain's Gold Medal of Sporting Merit.
Other leading figures of Spanish sports also expressed their condolences.
Barcelona midfielder Andres Iniesta extended his sympathies to her family while top-ranked tennis player Rafael Nadal said it was "very bad news for the world of sport in general, for the Spanish sport especially."
De Villota accident last year occurred while she was driving an F1 car for only the fourth time — and first for Marussia — and hit a support truck during a straight-line exercise near an airfield in England. An internal team investigation concluded the car was not at fault.
She first drove an F1 car in 2011, a Renault at the Paul Ricard circuit in Marseille, France.
Her death comes when de Villota seemed to be moving past her accident.
She told Hola magazine in February she felt "free" and "back to being me" after returning to driving on normal roads.
She returned to a F1 paddock for the first time in May at the Spanish GP. There she told the AP that she felt a mix of "adrenaline and also a little bit of sadness" on again being near the sport that almost cost her her life.
In July, she married boyfriend Rodrigo Garcia. She was active in charity work and a member of the FIA's women's commission.
On Monday, she was to present a book "Life is a Gift," detailing her ordeal following her driving accident.
AP Sports Writer Jim Armstrong contributed to this report from Suzuka, Japan.