Voters in the southwest city of Iwakuni, home to a US marine base, voted 8-1 on Sunday against bringing more aircraft and troops to the facility. The expansion is part of an agreement already reached between Tokyo and Washington.
The referendum vote is non-binding, but the result will complicate plans to finalise negotiations and the reorganisation of the nearly 50,000 US soldiers in Japan by the end of March.
Shinzo Abe, a government spokesman, said: "We will respect the result of the referendum, but on the other hand, we would like to reach an agreement in the Japan-US negotiations."
Junichiro Koizumi, the prime minister, said Tokyo would aim to meet the end-of-March deadline, but acknowledged that the plan faced stiff opposition from local communities.
"If a referendum were held, the result would be a 'No' vote anywhere. That is the difficulty with issues related to national security," he said.
Opposition from local communities concerned about noise, accidents and crime associated with US bases has hindered efforts to finalise the overall plan, part of Washington's global strategy to make its military more flexible.
National vs local interests
More than 43,000 residents voted to reject the plan while about 5000 were in favour, according to officials in Iwakuni, 1000km (600 miles) west of Tokyo.
The turnout was almost 59%, with 50% needed for the referendum to be valid, even though the government does not have to abide by it.
The Yomiuri Shimbun daily newspaper said central and local governments should hold talks to try to meet the interests of residents, but added that the realignment was aimed at dealing with changing security concerns, including North Korea's nuclear development and China's growing military.
The conservative paper said in an editorial: "Iwakuni should not cause confusion by uselessly opposing the government."
Situated 550km from the border between North and South Korea, US Marine Corps Air Station Iwakuni would play a role in any conflict involving the peninsula.
But Katsusuke Ihara, the mayor of Iwakuni, who called the referendum, said residents had the right to be heard.
"This is the voice of the people," Ihara told a private Japanese TV broadcaster late on Sunday.
"I have no right to comment on national issues such as security, but it is only natural for the people of Iwakuni to speak out on aspects that affect their lives."
People protest against the plans
for the US base
The Iwakuni referendum could add momentum to opposition in other communities, including the southern island of Okinawa, home to the bulk of the US forces in Japan.
Keiichi Inamine, the governor of Okinawa, reiterated on Sunday his opposition to a plan to relocate a marine heliport inside the prefecture.
Besides troop movements, Tokyo and Washington are also discussing how much Japan should pay to move 7000 marines to Guam from Okinawa.