The Brazilian angrily reacted to the Englishman's recent criticism of Neymar and the Brazil national team on social networking site Twitter, prior to Paris Saint-Germain's 2-2 draw with Barcelona in the Champions League on Wednesday.
Having labelled him a "FatBoy" on Tuesday, Barton reignited their feud on Wednesday morning.
"2 questions for Thiago Silva. Firstly, Why are YOU talking about ME, in your press conference before PSG v Barca? Very strange," Barton tweeted.
"Secondly, Are you Pre-Op or Post-Op? #transsexual #thiagosaladyboy.
"Baffles me, which way he's going. Is he a man changing to a woman or a woman changing to a man? Can't work it out."
Barton's loan spell with Marseille from Queens Park Rangers is due to expire in the summer.