The American flag was lowered at the US consulate in Chengdu on Monday and Chinese authorities entered the building as Beijing carried out a Cold War-style retaliatory closure of the mission.
Relations have deteriorated in recent weeks in an intensifying standoff between Washington and Beijing, with the Chengdu mission ordered to shut in retaliation for the forced closure of the Chinese consulate in Houston, Texas.
Beijing confirmed that the consulate had closed at 10:00 am (0200 GMT) on Monday.
"Afterwards, Chinese authorities entered through the front entrance and took it over," the foreign ministry said in a statement.
Both consulates closed 72 hours after the respective orders were made.
A State Department spokesman told AFP the mission's diplomats and their dependents would leave China by August 27.
Video footage published later Monday by the US embassy on Chinese social media platform Weibo showed US Marines in dress uniform lowering and folding the Stars and Stripes from the front of the consulate, before handing it to consul-general Jim Mullinax.
Diplomatic tensions have soared between the two powers, with both alleging the other had endangered national security.
Footage from Chinese state media showed officials arriving at the consulate in a minibus and walking into the front of the building.
They were followed by several workers in full hazmat suits and goggles, carrying cleaning equipment.
Two men wearing white gloves were shown covering up a US consulate sign on the wall outside the building with a large grey sheet.
A crowd gathered Monday outside the mission, and one man was escorted away by police after waving a banner and two Chinese flags while shouting "Long live China's Communist Party".
Over the weekend, removal trucks entered the site and cleaners were seen carting large black rubbish bags from the consulate.
AFP reporters saw workers removing the US insignia from the front of the building
"Today, we bid farewell to the US consulate in Chengdu. We will miss you forever," the US embassy in Beijing posted on Weibo.
In a separate statement, the consul-general posted that his mission had provided services to more than 200 million people in southwest China and Tibet, and had given "thousands" of visas every day to Chinese students and tourists.
"We are disappointed by the Chinese Communist Party's decision," a State Department spokesperson told AFP Monday.
The Chengdu consulate covered southwest China, including Tibet. Many Tibetans accuse the central government of religious repression and eroding their culture.
Beijing says closing the consulate was a "legitimate and necessary response to the unreasonable measures by the United States", and alleged that staff at the diplomatic mission endangered China's security and interests.
Foreign ministry spokesman Wang Wenbin told reporters that some US staff in the Chengdu consulate "were engaged in activities outside of their capacity, interfered in China's internal affairs, and endangered China's security and interests".
Washington officials, meanwhile, said there had been unacceptable efforts by the Chinese consulate in Houston to steal US corporate secrets and proprietary medical and scientific research.
Tensions have soared between the world's two biggest economic powers on a range of fronts including trade, China's handling of coronavirus and a tough new security law imposed on Hong Kong, with US officials warning of a "new tyranny" from China.
The last Chinese diplomats left the Houston consulate on Friday.
Beijing said Saturday that US agents "forcibly" entered the Houston consulate, which it said was "China's national property".
Its statement warned that "China will make a proper and necessary response in this regard".
The nationalistic tabloid Global Times warned in an editorial Monday that if Washington was "determined to push China-US ties in the worst direction... the 21st century will be darker and even more explosive than the Cold War era".
It said the rising tensions could lead to "unprecedented catastrophe".