Kenneth Boyd, 57, was pronounced dead after being executed by lethal injection at a prison in Raleigh, North Carolina.
The Vietnam war veteran, with a history of alcohol abuse, was condemned to death after being found guilty of the 1988 murders of his wife and father-in-law.
In the lead up to his death, about 100 anti-death penalty campaigners gathered outside the prison and read the names of the 999 other convicts executed in the US.
Up to 18 of them were detained and charged with trespassing after stepping onto prison property.
Boyd's family earlier said they did not expect a reprieve, and the condemned man was resigned to his fate.
His last chance of life ran out less than four hours before he was put to death when Governor Mike Easley said he did not find a compelling reason to grant clemency.
In his final few hours he ate a last meal of steak and Pepsi and met with his family for the last time.
His lawyer said he did not want to be remembered for being number 1,000.
"He said it best: I'm a person, not a statistic," lawyer Thomas Maher told Reuters.
Boyd was on death row in North Carolina for 11 years.
A photograph of North Carolina prisoner 0040519 shows a white man with a hollow face and fatigued eyes, framed by graying hair and a drooping moustache.
Protests
The Supreme Court ruled in 1976 that executions were allowable under the US constitution, which forbids cruel and unusual punishment.
"The 1,000th execution is a significant event in the nation's 30-year experiment with capital punishment," said Richard Dieter, director of the Washington-based Death Penalty Information Centre.
"But it is not indicative of an expanding or strongly endorsed use of capital punishment," Mr Dieter said.
"The impending milestone occurs at a time when the country is sharply moving away from the use of the death penalty."
North Carolina has conducted its executions at 2am since the penalty was reinstated, said Keith Acree, spokesman for the North Carolina Department of Corrections.
More than 3,000 people are currently on death row in US prisons.
But despite the bleak milepost, statistics show a 50 per cent decline in the number of death sentences since the late 1990s, and a 40 percent drop in executions since they peaked at 98 in 1999.
In the last 32 years, 122 death row inmates have been exonerated and released.
Last month, a Gallup poll showed that 64 percent of Americans remain in favour of capital punishment, down from 80 percent in the 1990s.
The death penalty is legal in 38 US states, but many seldom or never use it.
More than half of all executions take place in three states: Texas has executed 355 people, Virginia has put to death 94 and Oklahoma another 79, since 1976.
The US government also has the death penalty on its books for federal cases.
The most prominent federal execution in recent years was that of Timothy McVeigh, who was executed in 2001 for the 1995 bombing of an Oklahoma City US government building in which 168 died.
The first person executed after the Supreme Court's 1976 ruling was Gary Gilmore, who was killed by firing squad in Utah in 1977.
He was immortalised in American author Norman Mailer's "The Executioner's Song."