Gates' comments, coming after accusations by a State Department official, were the strongest by a Cabinet secretary about Iran's support of the terrorist group in Afghanistan.
Basing his conclusions on new intelligence, Gates said "given the quantities (of weapons) that we're seeing, it is difficult to believe that it is associated with smuggling or the drug business or that it's taking place without the knowledge of the Iranian government."
He said that the latest information indicates a "fairly substantial flow of weapons" is crossing into Afghanistan.
Tehran rejects the charge that it is aiding the Taliban and contends the accusation is part of a broad anti-Iranian campaign. Iran says it makes no sense that a Shiite-led government like itself would help the fundamentalist Sunni movement of the Taliban.
Undersecretary of State Nicholas Burns told CNN on Wednesday that "there's irrefutable evidence the Iranians are now doing this."
"It's certainly coming from the government of Iran," he said. "It's coming from the Iranian Revolutionary Guard corps command, which is a basic unit of the Iranian government."
State Department spokesman Sean McCormack told reporters "it certainly is hard to believe that the Iranian government isn't involved in some way, shape or form in this.
Gates and other defense officials would not go as far as Burns did. The Pentagon chief also said he was not as certain about the link to Iran's Quds Force, which is accused of arming and training Iraqi militants.
In April, Gen. Peter Pace, chairman of the Joint Chiefs of Staff, disclosed that Iranian-made weapons intended for Taliban insurgents were intercepted by U.S. forces in Afghanistan.
Pace said at the time that it was not clear which Iranian entity was responsible for the arms, which included mortars and C-4 plastic explosives.
Gates made his comments to reporters during a visit to Ramstein Air Base in Germany. He stopped at the base to visit injured troops and awarded six Purple Heart medals to wounded service members at the Landstuhl Regional Medical Center.
The secretary then went to Brussels, Belgium, for a meeting of NATO defense ministers.
Gates said he will press allies for more troops, trainers and equipment for Afghanistan.
He has been frustrated by NATO members' slow progress in meeting commitments in Afghanistan. But he said Wednesday that several countries recently indicated a willingness to increase the size of their force in Afghanistan or extend the length of their stay.
"I think countries are taking this seriously, and so I will continue to press in Brussels," Gates said at a news conference at Ramstein.
In February and again in April, Gates exhorted NATO allies to bolster their troop commitments in Afghanistan so the alliance could launch its own offensive against the Taliban and pre-empt what has been an annual spring increase in insurgent attacks.
That offensive got under way with the aid of additional U.S. troops.