Since winning the US election in November, President Donald Trump has refused to rule out the use of force to seize the Panama Canal, built by Washington over a century ago and later handed over to Panama.
Around 40 percent of US container traffic passes through the narrow body of water linking the Caribbean Sea with the Pacific Ocean.
Beyond the tolls paid, Washington has appeared chiefly concerned about Chinese investment in the 80-kilometre (50-mile) long canal, which handles five percent of global maritime trade.
The latest controversy over the Panama Canal comes at the end of Rubio's week-long visit to Central America, his first as the US top diplomat.
He had threatened action against Panama unless it made immediate changes to reduce Chinese influence on the canal.
Beijing on Friday said Rubio's remarks "unjustly accuse China" and "deliberately sow discord between China and relevant Latin American countries".
China has "lodged a solemn representation with the US side", an unnamed spokesperson added.
The country's President Jose Raul Mulino on Thursday confirmed that Panama had pulled out of the Belt and Road Initiative (BRI) infrastructure programme.
Rubio hailed that pledge as a "great step forward" for bilateral relations.
In response, Beijing said it "firmly opposes the US using pressure and coercion to smear and undermine Belt and Road cooperation".
"Its achievements have benefited people in countries such as Panama," foreign ministry spokesman Lin told a briefing.
The BRI is a massive infrastructure project that is a central pillar of Chinese President Xi Jinping's bid to expand his country's clout overseas.
Western critics accuse China of using the BRI to enmesh developing nations in unsustainable debt to exert diplomatic leverage over them or even seize their assets.
Beijing on Friday insisted that it "supports Panama's sovereignty over the canal".
"The US side's attacks and interference in relevant cooperation once again expose its hegemonic nature," the foreign ministry said.
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