While the length of the closure isn't clear, the U.S. already has started looking for supply line alternatives as it prepares to almost double its number of soldiers in Afghanistan next year.
A U.S. military spokesman in Afghanistan welcomed the operation against the extremists, despite the suspension of the supply route.
"We are glad that they're helping clean out what they call miscreants in that area that have been attacking the supply line," said Col. Greg Julian. "Temporary closure (of the supply line) is not a problem. It's best that they conduct this operation and clear out these trouble spots."
The road through the Khyber Pass in the northwest of Pakistan has come under increasing attacks by militants seeking to squeeze Western forces fighting a resurgent Taliban in Afghanistan in recent months.
U.S. and NATO soldiers in landlocked Afghanistan rely on the winding, mountainous route for delivery of up to 75 percent of their fuel, food and other logistical goods, which arrive in Pakistan via the port city of Karachi.
American commanders insist the attacks are not disrupting their mission in Afghanistan, but are exploring new routes. They also say they have enough supplies to last many weeks in the case the routes are blocked.
But the U.S. next year will send up to 30,000 new forces to the country to reinforce the 32,000 American soldiers already there, requiring even more support and supplies.
Last month, The Associated Press reported that NATO was close to reaching deals with Central Asian countries north of Afghanistan that would allow the alliance to truck in "non-lethal" supplies from there.
The road has been temporarily closed to traffic at least twice this year for security reasons, each time for no longer than several days.
Tariq Hayat Khan, top administration official in the Khyber area, said security forces were battling the militants using helicopter gunships and heavy artillery.
"This operation will continue until the goal is achieved, which is nothing less then the elimination of troublemakers," Khan told The Associated Press. He did not say how long the road would be closed for.
He said he had no information on any casualties.
Gunmen have staged a series of raids on truck depots near the Pakistani city of Peshawar in recent weeks, killing several guards and burning hundreds of vehicles, including dozens of U.S.-bought Humvees destined for the Afghan army.
During the summer, militants attacked and torched dozens of U.S. supply trucks on Afghanistan's main highway.
A smaller number of supplies arrive in Pakistan by a second land crossing at Chaman in the southwest. That road was open Tuesday, a witness said.
NATO says it is investigating other possible supply routes to Afghanistan — such as through central Asian states to its north. Guns and ammunition are not shipped through Pakistan.
On a normal day, some 300 trucks carrying military supplies travel up the pass. Media reports have said that truck companies are becoming increasingly unwilling to transport the goods because of the danger.