He is accused of conniving in a "secret plan" with foreign entities which began in 1993, according to a court statement.
Evidence of a conspiracy was substantial enough to charge the Cambodia National Rescue Party (CNRP) chief under the penal code section for "treason and espionage", it added.
Kem Sokha was arrested early Sunday in a swoop by hundreds of security forces at his home in the Cambodian capital.
Hours later strongman premier Hun Sen accused the politician of being in cahoots with the United States, although he provided no evidence in a typically bombastic speech.
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Hun Sen's opponents, NGOs and the critical press have been smothered by court cases and threats ahead of a crunch general election next year.
Hun Sen is determined to extend his three decade rule and withstand the burgeoning popularity of the CNRP.
Yesterday, The Cambodia Daily, one of the last independent newspapers in the kingdom, was shuttered by a tax claim it says is trumped up to muzzle its critical reporting.