According to the CVVM polling agency, 68 percent of respondents said they opposed the proposal, while 26 said they approved. In February, 61 percent said they opposed the idea in a similar poll.
The government-sponsored polling agency said it questioned 1,011 people on April 2-9, and said the margin of error for the survey was plus or minus three percentage points.
The U.S. wants to host a missile tracking radar system at the base, and is in talks with Poland to house 10 interceptor rockets on its territory as part of plans for a missile defense shield that Washington says would protect against a potential threat from the Middle East.
The Czech government supports the base, and has agreed to start negotiations with Washington. Opposition parties have demanded a national referendum on the issue.