"Our opposition to these sales is long and public. We believe it's unhelpful," Pentagon spokesman Colonel Steve Warren told reporters.
"We are raising that through the appropriate diplomatic channels," he said.
Earlier, Russian President Vladimir Putin signed a decree lifting a self-imposed ban on supplying the missile system to the Islamic republic.
In doing so, he pre-empted the possible future lifting of sanctions against Iran if it agrees on an accord to limit its nuclear program.
Warren said it was not immediately clear whether Putin's decision was in itself a violation of previously agreed international sanctions.
While not the most sophisticated of Russia's missile systems, the S-300 would bolster Iran's defenses against any attack on its nuclear sites.
Neither Israel nor the United States have ruled out air strikes if Tehran pursues what Western powers fear is a bid to develop a nuclear bomb.
Moscow blocked a previous planned delivery of the surface-to-air missiles to Tehran in 2010 after the United Nations slapped sanctions on Iran.
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