Rubiales announced his resignation in a statement in which he said his position had become untenable, with the Spanish Football Federation (RFEF) later confirming he had sent a resignation letter to acting president Pedro Rocha.
His kiss of player Jenni Hermoso after the World Cup win in Sydney on Aug. 20 had caused outrage among players, government officials and many in wider Spanish society and raised questions over sexism in sport.
His decision to go came after further blows to his position in the last few days.
A Spanish prosecutor filed a complaint with the High Court against Rubiales on Friday for sexual assault and coercion.
The complaint - which came after Hermoso opted earlier in the week to lodge a criminal complaint over the incident - describes how Rubiales kissed Hermoso on the mouth "without her consent" while holding her head with both hands after Spain defeated England to clinch the World Cup.
Rubiales has said the kiss was mutual and consensual, and had until Sunday defied calls from players, government officials and others for him to resign.
Rubiales had also been suspended for three months from all football activities by FIFA pending an investigation by soccer's world governing body into his actions.
"After the rapid suspension carried out by FIFA, plus the rest of the proceedings opened against me, it is clear that I will not be able to return to my position," Rubiales said in his statement.
"Insisting on waiting and clinging ... is not going to contribute anything positive, neither to the Federation nor to Spanish football. Among other things, because there are de facto powers that will prevent my return," he added.
He said he had also stepped down as a vice president of European soccer body UEFA. He posted his statement on X, the social media platform previously known as Twitter.
Reuters reached out to Hermoso's representative for comment on Rubiales' resignation, and they did not return the telephone call.
Hermoso, Spain's all-time top scorer with 51 goals, currently plays at Mexican top-tier side Pachuca, where she moved in June 2022 following her second spell at Barcelona.
Spanish government ministers, who had been solidly behind Hermoso and others calling for Rubiales to go, celebrated his departure.
"The feminist country is advancing faster and faster," Spain's acting Labour Minister Yolanda Diaz posted on X after news of Rubiales' resignation. "The transformation and improvement of our lives is inevitable. We are with you, Jenni, and with all women."
"It's over," said Spain's Equality Minister Irene Montero.
The Spanish Football Federation said in a statement that Rubiales had resigned as its president and also as a vice-president of UEFA.
"This has been made known to the federal entity through a letter to (acting RFEF president) Pedro Rocha Junco," the RFEF statement said.
Spanish women's players union FUTPRO did not immediately respond to Reuters' request for comment.
Although he stepped down as boss of the federation, Rubiales continued to defend his version of events.
A message posted with his statement on Sunday said, "I will defend my honour. I will defend my innocence. I have faith in the future. I have faith in the truth.".
In an interview with journalist Piers Morgan on Sunday Rubiales added he made the decision to resign having talked with family and friends.
"Luis, you have to focus on your dignity and continue your life. (Otherwise) you will probably be hurting more people you love and the sport you love," he quoted them as saying.
