Treason is a capital offence in Uganda and if found guilty the 68-year-old cold be sentenced to death.
He was charged alongside two other suspects, but they did not enter a plea on the grounds that the charges against them could only be heard in a higher court.
Besigye, who has run for president against long-serving leader Yoweri Museveni four times, has been in detention since he was dramatically abducted in Kenya in November and taken back to Uganda to face a military trial.
But a landmark ruling by the Supreme Court last month said that trying civilians in military courts was unconstitutional and ordering all such cases to be transferred.
The move angered President Museveni, who called it “a wrong decision”.
At the start of last week, Besigye had begun a hunger strike over his continued detention.
The charges stem from accusations that he was plotting to remove Museveni from power by force.
Friday was the first first time Besigye had appeared before a civilian court for formal charges, after the Supreme Court ruling.
Visibly frail, he was wheeled before the Nakawa magistrate court in the capital, Kampala, alongside his aide and co-accused Obeid Lutale.
The opposition politician has now called-off his hunger strike, his lawyer Erias Lukwago told journalists.
According to the charge sheet presented before court, Besigye is accused of holding meetings in Switzerland, Greece and Kenya between 2023 and November last year in a plot to overturn the government.
He was also accused of soliciting for military, financial and other logistical support to topple Museveni’s government.
Besigye was charged alongside Mr Lutale and Denis Oola, a military officer.
Previous charges heard in the military court of illegal possession of ammunition and treachery were not included in the new charge sheet.
During proceedings, Besigye’s lawyer asked the court to order that he be transferred to a better health facility outside the prison but Magistrate Esther Nyadoi dismissed the application, saying she was unable to grant such a request.
She ordered the accused to be remanded until next month to allow the prosecutor to finish the investigation.
Besigye used to be a personal doctor for Museveni, who has been in power since 1986.
The veteran opposition figure has previously accused the Ugandan authorities of political persecution.
He has been less active in politics in recent years, and did not contest the 2021 election.
Source: bbc.com
