The pop superstar's song God Control calls for people to "wake up" about the reality of gun violence in the US.
The scenes in the music video recall the 2016 Pulse nightclub shooting in Orlando, Florida, where 49 people were killed.
Pulse survivor Patience Carter said she was "truly disturbed" by the video.
The eight-minute video shows actors playing revellers being shot and lying lifeless and covered in blood in a fictional nightclub called The Globe.
"I couldn't even watch after the first 45 secs @Madonna," Carter wrote on Twitter.
"There are so many creative avenues that could've been taken to bring awareness to gun control. The victims of these mass shootings should always be taken into consideration. I applaud the attempt, but I am truly disturbed."
Another survivor, Brandon Wolf, wrote: "Appreciate the message, but please remember that there are people behind the prop you're using."
The singer told CNN her response to those who had a problem with the graphic violence in the video was that "this is what happens when people shoot".
She told People: "Seeing the reality, and the brutality of things makes you wake up. This is really happening. This is what it looks like. Does it make you feel bad? Good, 'cause then maybe you will do something about it."
She also said gun violence "is the biggest problem in America right now", adding: "I cannot take it any more."
Actor George Takei, who set up the anti-gun group 1Pulse4America after the Orlando tragedy, was among those to praise the singer.
March For Our Lives, the lobby group founded after the 2018 Parkland school shooting, also thanked the pop star.
The Guardian's music editor Ben Beaumont-Thomas said the video would be "very triggering for anyone disturbed by the Pulse nightclub terror attack", but added Madonna was making her voice heard about gun reform "louder and clearer than almost anyone in pop culture".