The 66-year-old co-founded the now-banned Cambodia National Rescue Party -- once considered the sole viable opponent to the ruling Cambodian People's Party (CPP) led by strongman premier Hun Sen.
But Kem Sokha was arrested in 2017 and his party dissolved ahead of widely criticised elections the following year -- leaving the CPP to run virtually uncontested.
The opposition leader was detained first in a remote prison then placed under house arrest before his bail conditions were relaxed in November last year.
He stands accused of conspiring in a "secret plan" with foreign entities to overthrow the government, according to court documents -- charges he has repeatedly denied.
Hun Sen, Asia's longest-serving leader, has ruled Cambodia with an iron fist for 35 years.
Hundreds of police officers surrounded Phnom Penh Court Wednesday morning as Kem Sokha arrived in a green SUV and was ushered quickly into the courthouse before journalists could speak to him.
Reporters and human rights monitors were not allowed to attend the hearing due to the size of the venue, a court official told AFP, with the limited seating reserved for foreign diplomats and relatives.
Kem Sokha's daughter Kem Monovithya on Wednesday decried the proceedings.
"This whole ordeal is a farce," she told AFP.
"It is damaging to Cambodia's image. We hope he will be acquitted, so Cambodia can begin to get back on a democratic path," she said.
Amnesty International called the trial "a mockery of justice".
"The non-existent crime was politically manufactured to further the suppression of the opposition party," said regional director Nicholas Bequelin in a statement.
The US State Department said the charges "appear to be politically motivated".
If convicted, Kem Sokha faces up to 30 years in jail.
Due to concerns over human rights, the European Union is reviewing whether Cambodia should be withdrawn from a scheme allowing it to export goods other than weapons to the EU tariff and duty-free.
If axed, it could deal a blow worth billions to the kingdom's lucrative garment sector.
The government might attempt a "compromise" to relieve international pressure, political analyst Ou Virak told AFP.
This could come in the form of a royal pardon if Kem Sokha is convicted, he said.