An Indian fisherman was killed and three others were wounded on Monday when a US navy ship fired at their small boat off Dubai in the tense waters of the southern Gulf, officials said.
US defence officials said the motorboat had ignored warnings not to approach the refuelling ship USNS Rappahannock, and that sailors on board the American vessel feared it could pose a threat.
"Since 2000, we've been very concerned about small boats," a defence official in Washington said, referring to the year of a deadly suicide bomb attack against the destroyer USS Cole in the Yemeni port of Aden.
A United Arab Emirates official said one fisherman was killed and three other Indians were wounded.
"The services concerned are now investigating this incident," foreign ministry official Tareq Amed al-Hidan said, quoted by state news agency WAM.
A statement from the US Fifth Fleet, which is based in Bahrain and on alert for possible Iranian action in Gulf waters, said the crew had opened fire as a last resort.
"An embarked security team aboard a US navy vessel fired upon a small motor vessel after it disregarded warnings and rapidly approached the US ship near Jebel Ali," it said, referring to an Emirati port city.
"The USNS Rappahannock used a series of non-lethal, preplanned responses to warn the vessel before resorting to lethal force," it said.
"The US crew repeatedly attempted to warn the vessel's operators to turn away from their deliberate approach," it added.
"When those efforts failed to deter the approaching vessel, the security team on the Rappahannock fired rounds from a .50-calibre machine gun."
The US navy has been building up its forces in the oil-rich region amid mounting tensions with Iran over its controversial nuclear programme.
Tehran has warned it could close the Strait of Hormuz in the southern Gulf if international sanctions begin to bite, potentially disrupting shipping and world oil supplies through the strategic waterway.
Washington has deployed two aircraft carriers to the region - the USS Abraham Lincoln and the USS Enterprise - and doubled its minesweeper fleet in the area from four to eight ships on June 23.
And on Monday, the Pentagon confirmed that it had brought forward the deployment off a third strike group, led by the carrier USS John-Stennis, by four months in order to further bolster its presence.
The deployment aims to warn off Iran over its threats to mine the narrow strait through which about a fifth of the world's traded oil passes.
In October 2000, 17 US sailors were killed when militants in an explosives-laden skiff blew a 30-by-30-foot (10-by-10-metre) hole in the USS Cole in Aden. Al-Qaeda claimed responsibility for the attack.