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The Northern Irishman had a one-under-par 71 in his second round on Friday but was five over par overall after a disastrous 78 in the first round.
The four-times major winner ended four shots off the cut mark at Erin Hills, as did compatriot Graeme McDowell, who added an imoroved 73 to his opening 76.
McIlroy was playing just his seventh tournament of 2017 due to a rib injury.
He won the US Open with a record 16-under-par total in 2011, but missed the cut at Oakmont last year following rounds of 77 and 71.
The 28-year-old needed to make a fast start on Friday to avoid another early exit, but missed from five feet for birdie on the 10th - his opening hole - and 12 feet on the 11th, before a poor chip from the back of the 12th green led to a bogey.
McIlroy bounced back with a birdie from 12 feet on the 13th, but carded a hat-trick of bogeys from the first to slump to nine over par.
McIlroy rallied with four birdies in his six closing holes but it was not enough to keep him in the tournament.
"I shot myself in the foot yesterday and gave myself too much to do," he said afterwards.
McDowell, the 2010 US Open champion, carded birdies at the first and second holes but his card was marred by three bogeys.
Irishman Shane Lowry did make the cut and is tied for 55th place on one over after a second round of 74 on Friday.
Paul Dunne failed to make the weekend however as he finished four over after rounds of 75 and 73.
English pair Paul Casey and Tommy Fleetwood head the field on seven under, along with Americans Brian Harman and Brooks Koepka.
Rickie Fowler, Jamie Lovemark and JB Holmes lie one shot behind the leading quartet.
World number two Rory McIlroy has missed the cut at the US Open for the second year running.