Manchin's office did not immediately comment on the NRA ad.
The NRA plans to spend $100,000 airing it in West Virginia markets over the next two weeks, spokeswoman Jacqueline Otto said in a statement.
The killing of 20 first-graders and six adults at Sandy Hook Elementary School in Newtown, Conn., by a gunman six months ago this Friday helped spur Manchin's legislation.
Some of the victims' families vowed to keep up the pressure for stricter gun control as they met with the lawmakers in Washington.
"This mother's heartbreak that I carry, this life sentence that I have, no one should ever have to bear this burden," said Nelba Marquez-Greene, whose daughter, Ana, was among the Sandy Hook victims.
Newtown families also met for nearly an hour Wednesday with House Speaker John Boehner, R-Ohio, and others in the House, where leaders have expressed interest in strengthening mental health programs but not in expanding background checks.
"I thanked them for their courage and willingness to come forward and talk to us," House Majority Leader Eric Cantor, R-Va., said. "I know that they're continuing to seek how to solve the problem and we're going to continue to go and to listen to them and work" on the issue, he said.
Representing a state where gun ownership and hunting are long-held traditions, Manchin has said he believed he could credibly provide reasonable proposals to the resulting debate.
The NRA ad invokes a much-discussed TV spot from Manchin's 2010 Senate campaign.
Called "Dead Aim," it shows Manchin loading a rifle as he touts his NRA backing and support of the Second Amendment. He then shoots environmental legislation sought by the Obama administration. Manchin won that special election to complete the term of the late Robert C. Byrd and was elected to a full six-year term in 2012.
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