Republicans hailed it as a hard-fought victory, saying the bill -- which also cuts funding to women's health care provider Planned Parenthood -- highlights sharp policy differences between their party and rival Democrats during the 2016 presidential election race.
Obama is certain to veto the measure, which passed the Senate last month under special rules that prevented Democrats from blocking it.
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It was the first major congressional vote of 2016, coming just two months after Paul Ryan became the new Speaker of the House.
And yet it is the latest in a long line of symbolic votes that Republicans have held in recent years, frustrating millions of conservatives across the American heartland who have grown angry with ineffectual Republican leaders in Congress.
"For the first time in five years, we will finally put a bill on the president's desk that defunds Obamacare," Ryan told reporters, noting that Democrats have been "blocking and filibustering these bills" for years.
"We are confronting the president with the hard, honest truth: Obamacare doesn't work," he added.
Obama was seeking to talk about "anything but his failures," but Republicans were "not going to let him take a soft course," he said.
"We need to make this year about ideas, not about Obama's distractions."
Democrats dismissed the vote as the 62nd attempt by Republicans to repeal, defund or otherwise dismantle the Affordable Care Act that narrowly passed a Democrat-led Congress in 2010.
"I don't understand their obsession" with repealing the health law, House Democrat Jim McGovern said, noting that doing so would "throw 22 million people out of health insurance plans."
As for the law putting a one-year moratorium on federal funding for Planned Parenthood, Democrats warned it would deny health care access to millions, particularly impoverished women.
"It's cruel. It's a cruel thing to do," McGovern said.
Debate over Planned Parenthood exploded last year, when anti-abortion activists released secretly-recorded videos showing the organisation's officials discussing use of aborted fetal tissue for medical research.
Conservative critics, many of whom seek to outlaw abortion in the United States, have accused the organisation of selling fetal organs and body parts for profit, and encouraging women to have abortions in order to expand such operations.
Seeking to douse the months-long scandal, Planned Parenthood announced in October that it will no longer accept reimbursements for costs of the donations.
House Republican Ann Wagner called the vote "a victory for women's health."
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