Hichilema, leader of the United Party for National Development (UPND), has been in custody since April over an incident.
Hichilema, leader of the United Party for National Development (UPND), has been in custody since April over an incident when he allegedly failed to give way to President Edgar Lungu's motorcade.
Lungu, who narrowly beat Hichilema in last year's presidential election, has dismissed allegations of growing authoritarianism and has accused his rivals of trying to overturn the election result.
Hichilema and five aides "denied the charge of treason and the state has decided to take the matter to Wednesday for trial," UPND spokesman Charles Kakoma told foreign journalists outside the court.
Police officers in riot gear sealed off the court precinct as scores of UPND supporters waited outside.
Kakoma added that Hichilema, who remained in custody, appeared in good health at the brief hearing.
A UPND lawyer, who declined to be named, had said on Sunday that the party expected the charges to be dropped and for Hichilema to walk free at the hearing.
President Lungu invoked emergency powers in July, increasing police powers of arrest and detention after he blamed opposition parties for a string of arson attacks.
Zambia has enjoyed relative stability since its first multi-party election in 1991.
But last year's election was marked by clashes between supporters of Lungu's Patriotic Front (PF) party and the UPND.
Hichilema, 55, says the vote was rigged and has refused to recognise Lungu as the president.
Parliament has suspended 48 UPND lawmakers after they boycotted an address by Lungu in March.
Police last week released the leader of a smaller opposition party who is a fierce critic of the president after one week in detention.
Savior Chishimba, leader of the United Progressive Party (UPP), was detained by plain-clothed police, triggering further accusations of a crackdown on dissent.
The government has also increased pressure on media outlets that support the opposition, eroding Zambia's reputation as a stable democracy.
Hichilema was arrested after allegedly putting Lungu's life in danger when his convoy failed to make way for the presidential motorcade in a high-speed road drama caught on video camera.
The two men were both travelling to Western province for a traditional ceremony.
Days later, more than 100 armed police surrounded Hichilema's house outside Lusaka, firing tear gas before detaining him and his aides.
Businessman-turned-politician Hichilema has claimed that he was assaulted by police during his arrest and has suffered mistreatment in detention.
Treason is a non-bailable offence in Zambia, with a minimum jail term of 15 years and a maximum sentence of death.
When he was arrested, Amnesty International said that Hichilema and the five other accused were "victims of longstanding persecution" by the authorities, and faced charges that are designed to "harass and intimidate".
Lungu did not mince his words during the election campaign, warning political rivals and activists that "if they push me against the wall, I will sacrifice democracy for peace."