Officials said the singer could not attend Thursday’s show because of unspecified “production issues”.
Lopez was due to perform alongside rapper Pitbull and Brazilian singer Claudia Leitte, with whom she has recorded the song We Are One (Ole Ola).
Pitball and Leitte will go ahead and perform accompanied by drummers Olodum.
“For production issues, Jennifer Lopez, one of the artists of the official song We Are One, will not be able to perform at the Fifa World Cup opening ceremony,” said a statement.
The 25-minute ceremony in Sao Paulo will feature 600 artists, including acrobatic gymnasts, trampolinists, martial arts-style performers and stilt walkers.
Last week, Lopez – whose hits include On The Floor and Jenny From The Block – said she was “thrilled” to be given the chance to perform at the opening ceremony.
“This is an amazing celebration of global unity, competition and sport,” she said.
Pitbull appeared not to be downhearted at Lopez’s withdrawal from the show.
“To be performing We Are One to the world, especially in such a beautiful country like Brazil, is going to be a lot of fun,” he said in a statement released by Fifa.
“But more than this, it is going to show the world that music is the universal language.”
Lopez also missed the January event where Pitbull and Leitte unveiled plans for the song alongside Fifa officials in Rio de Janeiro.
More than 60,000 people are expected to be in Sao Paulo’s Corinthians Arena to watch the ceremony and kick-off match between Brazil and Croatia.
Up to a billion people are expected to tune in on TV worldwide
Lopez’s cancellation comes amid protests and strikes in Brazil, where some are unhappy about the amount of money being spent on the tournament.
The action has been on a smaller scale to the mass protests last year, which delayed the building of stadiums and key infrastructure projects.
However, a poll by the Pew Research Centre suggested that 61% of the public now felt hosting the World Cup had been a bad idea, as it diverted money from public services such as healthcare.