The National Rifle Association announced its largest ad buy of the 2016 presidential race on Wednesday, promising $6.5 million in new spending to back Donald Trump and cementing the gun group as the GOP nominee’s staunchest outside ally.

One new 30-second ad suggests electing Democratic candidate Hillary Clinton would leave women “defenseless” as victims of crimes.

“Every woman has a right to defend herself with a gun if she chooses,” says Kristi McMains, an Indiana woman who pulled a gun to fend off a would-be attacker. “Hillary Clinton disagrees with that. Don’t let politicians take away your right to own a gun.”

The new ad is part of a blitz in Ohio, Pennsylvania, North Carolina and Virginia, as well as national cable and satellite.

Mr. Trump received the endorsement of the gun rights group at its annual convention in May, and has repeatedly touted his pro-gun bona fides on the trail, saying recent attacks such as the ones in Orlando and San Bernardino could have been mitigated if there were more guns in the room and bullets had been flying in the other direction.

And as other conservative-leaning groups like the U.S. Chamber of Commerce and the Koch brothers network shy away from actively engaging in the presidential race, the NRA has been there to try to pick up the slack.

The group has put more than $17 million into TV ads this cycle supporting Mr. Trump and attacking Mrs. Clinton, and that total doesn’t include digital and mail efforts, as well as other grass-roots activities.

“If ever there were a time to do everything possible to protect the Second Amendment, this is it. If elected, Hillary Clinton would work to eliminate our constitutional freedoms,” Chris W. Cox, executive director of the NRA’s legislative lobbying arm, said last month.

Gun control advocates say they’re prepared to fight back.

Everytown for Gun Safety, a gun control group co-founded by former New York City Mayor Michael Bloomberg in 2014, called the NRA’s latest ad a “special brand of women-targeted fear-mongering.”

Bucking conventional political wisdom, Mrs. Clinton is running on a fervent gun control platform this year. Sen. Tim Kaine, her running mate, said at Tuesday’s vice presidential debate that while he supports efforts like tighter background checks, he is a gun owner and a “strong Second Amendment supporter.”

Neither Mrs. Clinton nor Mr. Kaine has historically been all that good on guns, said Philip Van Cleave, president of the Virginia Citizens Defense League. But Mr. Van Cleave also said Democrats’ focus on gun control during the 2016 election is unprecedented.

He said he was shocked at Mr. Kaine’s claim during the debate that he was a Second Amendment supporter.

“I thought he was trying to make a joke,” Mr. Van Cleave said.

Mr. Kaine was governor of Virginia during the 2007 Virginia Tech massacre, and has described it as the worst day of his life. He and state lawmakers pushed for action on guns and the state’s mental health system in the aftermath of the attack.

He also vetoed legislation that would have let Virginians carry guns in their glove boxes and legislation to allow people to carry guns in bars.

“Neither one of them were ever good on guns, I don’t think. But this is the first time I’ve seen a platform like this where the Democrat Party and the people running are all running on a gun control platform that’s actually a key leg,” Mr. Van Cleave said. “I’ve never seen that before. And it’s disgraceful.”

Mr. Kaine has been “perennially ‘F’ rated” by the NRA at both the state and federal levels, the gun rights group said in a Twitter post Wednesday, to which Mr. Kaine replied: “And proud of it.”

Mr. Trump did raise some eyebrows in June when he said he planned to meet with the NRA to talk about preventing people on no-fly or terror watch lists from buying guns — a legislative initiative that congressional Democrats have been pushing for some time.

At last month’s first debate, he suggested it might be one area where he and the NRA may be at odds.

But the NRA says his position aligns with that of the gun rights group, which favors the approach of Sen. John Cornyn of Texas that would require a period of judicial review before a known or suspected terrorist is finally rejected from buying a gun.

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