Williams sent social media and the tennis world into frenzy on Thursday (AEST) when she posted a photo of herself on Snapchat with a caption that read simply "20 weeks" before the 23-time major winner deleted the post.
However, the 35 year-old American's representative confirmed the pregnancy in a widely circulated statement hours later.
"I'm happy to confirm Serena is expecting a baby this fall," the statement read.
Williams - who boasts an 8-1 win-loss record in 2017 - was due to make a WTA Tour comeback from a knee injury at the Madrid Open in May, having been sidelined in Indian Wells and Miami, but reports later suggested she will sit out the remainder of the season.
The legendary tennis player announced her engagement to Alexis Ohanian, the co-founder of Reddit, in December.
She won the Australian Open in January, beating sister Venus for her seventh title at Melbourne Park. The "20 weeks" timeline suggests Serena was pregnant at the time.
That victory also marked an Open-era record of 23 grand-slam titles for Serena.