Chair umpire Carlos Ramos penalised the tennis champion for receiving illegal coaching from the player box, People reports.
Williams wasn’t taking the accusation lying down and fired back at the umpire, "I don’t cheat to win, I’d rather lose. I’m just letting you know."
However the 39-time grand champion wasn’t done there and went on to call Ramos a “thief”.
"You will never, ever, ever be on another court of mine as long as you live," she said.
"You are the liar. When are you going to give me my apology? You owe me an apology. Say it. Say you’re sorry. And you stole a point from me. You’re a thief, too!"
Williams then refused to shake Ramos’ hand after the match was over.
Williams went down to Osaka 6-2, 6-4, however her outburst overshadowed the young star’s first-ever grand slam win.
"Well, I don’t want to be rude, I don’t want to do questions," Williams said in her on-court interview. "[Osaka] played well, this is her first Grand Slam. I know you guys were here rooting. Let’s make this the best moment we can. Let’s not boo anymore. We’re gonna get through this and we’re gonna be positive. No more booing!"
She then went on to further detail the outburst in her post-match interview, where she claimed the umpire’s remarks were sexist. A claim a number of other plays - both male and female - have backed up.
“I’m here fighting for women’s rights,” she said.
“The fact that I have to go through this is just an example for the next person that has emotions and wants to express themselves, and they wanna be a strong woman and they’re going to be allowed to do that because of today.
“Maybe it didn’t work out for me, but it’s going to work out for the next person.”
However the fall-out from the outburst didn’t end there, with Williams being fined $US17,000.
US tennis great Billie Jean King took to Twitter to applaud the star for speaking out as well as a number of other stars.