"Why believe in a god? Just be good for goodness' sake," reads the ad, which appeared Tuesday in the Washington Post and New York Times dailies and will feature on public transportation buses in Washington starting next week.
The aim of the advertisement is to "reach out to people who don't believe, to say that they're not alone, even during the holidays when they are being bombarded with religious messages," Roy Speckhardt, executive director of the American Humanist Association, which is behind the ad, told AFP.
Although the ad has had limited exposure through the two newspapers, it has already sparked reactions and lively online debate.
"It's OK not to believe, but don't expect eternal life at the end of time when Jesus returns," commented someone with the username Kassiani on the website of a Washington television station that ran a story about the ad.
An angry Daffoje wrote: "When we, as a nation begin to allow atheists, homosexuals and anyone who forms opinions to shape our nation, above the people and leaders who have great wisdom, clarity and understanding, we have stooped to an all-time low."
Others defended the right to free speech, while another user pointed out that Americans already avoid using the word Christmas, preferring instead to refer to the "holiday season" to avoid offending non-Christians.
Christianity is the dominant religion in the United States, with Protestants and Roman Catholics together making up around three-quarters of the population, according to a survey of 35,000 US adults conducted earlier year by the Pew Forum on Religion and Public Life.
Nearly six in 10 Americans pray every day, and around three-quarters believe in heaven as a place where people who have led good lives are rewarded, the study showed.
Oddly, one in five Americans who identified themselves as atheists in the survey also said they believe in God.