Mr Spicer made the comments at a daily news briefing about the April 4 chemical weapons attack in Syria that killed 87 people.
Washington has blamed the attack on the government of Syrian President Bashar al-Assad.
He said: "You had someone as despicable as Hitler who didn't even sink to using chemical weapons."
The Nazis murdered six million Jews during World War Two.
Many were killed in gas chambers in European concentration camps.
When asked by a reporter to clarify his comments, he said: "I think when you come to sarin gas, there was no, he was not using the gas on his own people the same way that Assad is doing."
The comments, made during the Jewish holiday of Passover, sparked outrage on social media and from some Holocaust memorial groups who accused him of minimising Hitler's crimes.
"Sean Spicer now lacks the integrity to serve as White House press secretary, and President Trump must fire him at once," said Steven Goldstein, executive director of the Anne Frank Centre for Mutual Respect.
Mr Spicer later emailed a statement to reporters who were asking for more explanation.
It read: "In no way was I trying to lessen the horrendous nature of the Holocaust. I was trying to draw a distinction of the tactic of using airplanes to drop chemical weapons on population centers. Any attack on innocent people is reprehensible and inexcusable.”
The U.S. Holocaust Museum did not mention Spicer's comments directly, but sent out a tweet shortly after the briefing that showed graphic footage of dead bodies U.S. forces found while liberating the Buchenwald concentration camp.