Hichilema, who has been held in custody for more than six weeks, will be detained until at least June 12, when the case will return to court.
Amnesty International said Hichilema and five others from the United Party for National Development (UPND) were victims of a "cynical ploy to silence all political opposition in Zambia".
Hichilema was arrested for allegedly endangering President Edgar Lungu's life when Hichilema's convoy failed to make way for the presidential motorcade on a main road.
Treason suspects are not eligible for bail in Zambia and could theoretically face the death penalty if found guilty.
"The Zambian authorities must stop misusing the criminal justice system to target and harass the political opposition," Amnesty said.
South Africa's main opposition party leader Mmusi Maimane led a demonstration in Pretoria today to support Hichilema.
Maimane, head of the Democratic Alliance, was barred from entering Zambia after he landed in Lusaka airport yesterday to attend the hearing.
He has since refused to recognise Lungu as president and has challenged the narrow poll defeat in court.
Outside the court today, Hichilema's supporters called for him to be released.
"He should have been president, but we have a dictator persecuting him," protester Stephen Katuta told AFP.
The election campaign was marked by clashes between supporters of Lungu's Patriotic Front party and Hichilema's UPND.
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