"We oppose any unilateral decision by either China or Taiwan to change the status quo," Bush said in Washington when asked about a proposed 20 March referendum in Taiwan.
"And the comments and actions made by the leader of Taiwan indicate that he may be willing to make decisions unilaterally to change the status quo, which we oppose," the US president added, seated along with visiting Chinese Premier Wen Jiabao.
Chen has argued that his proposed referendum falls short of any moves to change the status quo on sovereignty between his nationalist island and the mainland, which regards Taiwan as a renegade province.
But Bush's strong words make it clear that Washington does not agree.
The US, while a strong supporter of democratic Taiwan, which it is bound by law to defend, grants diplomatic recognition only to Beijing under the one-China policy.
Earlier, China warned Taiwan that it could "absolutely not accept" any moves towards independence.
Premier Wen Jiabao used White House talks on Tuesday with President George Bush to deliver the stark warning.
He emphasised China's view that the referendum was part of a separatist agenda in Taiwan.
"We very much appreciate the position adopted by President Bush toward the latest moves and developments in Taiwan"
"The Chinese government respects the desire of people in Taiwan for democracy," Wen said.
"But we must point out that the attempts of Taiwan authorities, headed by Chen Shui-bian, are only using democracy as an excuse and (an) attempt to resort to (a) defensive referendum to split Taiwan away from China.
"Such separatist activities are what the Chinese side can absolutely not accept and tolerate."
But Wen said as long as a "glimmer of hope" remained, China would try to settle the dispute peacefully.
"Stability can only be maintained through unswerving opposition and firm opposition to pro-independence activities," he said.
"We very much appreciate the position adopted by President Bush toward the latest moves and developments in Taiwan." Wen said.
Chen, leader of the pro-independence Democratic Progressive Party (DPP), said on Sunday that the "anti-missile, anti-war" referendum was aimed at asking China to dismantle hundreds of ballistic missiles targeting the island.