In a virtual carbon copy of last month's CONCACAF Gold Cup semi-final - where the American women also defeated Canada on penalties - Naeher saved three spot kicks and scored another to seal the host nation's victory in Columbus.
An entertaining battle went to a shoot-out after finishing deadlocked at 2-2 after 90 minutes and stoppage time.
The US looked to be heading for victory in regulation after two second-half goals from Sophia Smith had fired the hosts into a 2-1 lead after Adriana Leon's first-half opener for Canada.
But the Canadians were given a lifeline in the 84th minute when veteran defender Crystal Dunn bundled over Leon in the area.
Referee Crystal Sobers pointed to the spot and Leon coolly stepped up to bury the penalty and set up the shoot-out drama.
Olympic champions Canada seized the early initiative in the shoot-out, taking a 2-0 lead after Jessie Fleming and Leon scored either side of a miss from Trinity Rodman.
But Smith's penalty got it back to 2-1 and then Naeher saved from Jade Rose and Cloe Lacasse and scored her own penalty to help put the Americans in the driving seat.
Lindsey Horan's spot-kick made it 3-2 but Canada's Julia Grosso leveled to make it 3-3.
That left Emily Sonnett with the chance to seal victory, but the American midfielder blazed her penalty over the bar to set up sudden death.
Naeher saved from Canada substitute Evelyne Viens and Emily Fox duly rolled in the winning US penalty.
Earlier, Canada had taken the lead just before half-time when a defensive lapse allowed Leon to stroke home the opening goal in the 40th minute.
But Smith equalized on 50 minutes with a crisp low shot that flew into the bottom corner past Canada goalkeeper Kailen Sheridan.
The 23-year-old Portland Thorns forward then put the US 2-1 up on 68 minutes, rounding off a superb counter-attack and tucking away a low first-time finish after Rodman's perfectly weighted pass.
The hosts appeared to be heading for victory but Canada's late penalty forced a shoot-out.
The game was the last under US interim head coach Twila Kilgore, who will make way for Chelsea coach Emma Hayes later this year ahead of the Olympics.
