They are accused of killing Christopher Howes and his interpreter, who were kidnapped in March 1996 while clearing mines near Angkor Wat, the 12th century temple complex in the country's northwest.
The five defendants, among them Khem Ngun, a former guerilla leader who joined the Cambodian army in 1988 as a major general, appeared in a Phnom Penh court on Friday.
They have been charged with premeditated murder and illegal confinement of persons, and face life imprisonment if found guilty.
Witnesses claim Khem Ngun gave the order to kill the men.
Howes, a former soldier, was kidnapped along with his Cambodian mine-clearing team.
He later persuaded the guerrillas to free his colleagues, while holding him and his Cambodian interpreter hostage.
Two years after the incident, Scotland Yard investigators said they had firm evidence that the two men were taken to the then Khmer Rouge stronghold of Anlong Veng, near the Thai border, and later killed.
At the time of the first arrests in November last year, Howes' father, Roy, who lives in Backwell in southwest England, said his family would never recover from the incident.
"These people have wrecked my family," said Roy. "The pain is permanently with us."