The DJ revealed that he is still angry with the star's reference to "Molly" (ecstasy) at the Ultra electronic dance music festival earlier this year.
"You want to be 'hip' and 'cool' and 'funky grandma'? Fine. It's not my place to say you're irrelevant. If you're gonna come into my world, at least do it with a little more dignity," Deadmau5 told Rolling Stone.
"I understand she has millions more fans and is way more successful than I'll ever be. But it's like talking about slavery at a f**king blues concert. It's inappropriate."
Deadmau5 also made a pop at fellow DJ David Guetta, claiming that the French star doesn't always perform live at his shows.
"David Guetta has two iPods and a mixer and he just plays tracks - like, 'Here's one with Akon, check it out'," he said.
Deadmau5 will headline the main stage at this year's Wireless in London's Hyde Park on Friday, July 6.
Watch the music video for Madonna's 'Girl Gone Wild' below: