Clinton's 60-second ad, released Tuesday, uses video of Trump's speech in Baltimore on Monday, when he slammed Clinton's "deplorables" remark and called on her to retract it or drop out.
"You can't lead this nation if you have such a low opinion for its citizens," Trump said during his speech at the National Guard Association's annual conference.
That quote bookends the Clinton video, which highlights video of Trump calling Rosie O'Donnell "a disgusting pig" and referring to multiple individuals as "losers." 
"How stupid are the people of the country?" Trump asks in one video clip in the ad.
"We're building a wall. He's a Mexican," Trump says in another video.
The spot will run on national cable television, according to Clinton's campaign.
"If you won't say the KKK is deplorable, you have no business running the country," Clinton tweeted while sharing video of Pence, who did disavow Duke's support.
Clinton over the weekend said she regretted generalizing about Trump's supporters, while her campaign has defended its criticism of an attack on white nationalists supporting Trump.
Trump invited supporters on stage at a rally Monday night to denounce Clinton's comment and explain their support for the Republican presidential nominee.