Shrien Dewani, 34, also denied paying three hitmen to kill his wife Anni on their honeymoon in November 2010 at Western Cape High Court.
Through defence lawyer Francois Van Zyl, Dewani said he has "had sexual interaction with both males and females".
"I consider myself to be bisexual," the court was told.
"My sexual interactions with males were mostly physical experiences or email chats with people I met online or in clubs, including prostitutes," Dewani's witness statement said.
A German male prostitute is expected to be a key witness in the case and will tell the court that Dewani paid him for sex in the months leading up to the shooting, telling him he regretted his engagement with Anni but was too ashamed to back out due to family pressure, Independent newspaper reported.
Anni's father, Vinod Hindocha, is in Cape Town with his wife and other family members from Sweden for the trial.
Dewani was extradited from the UK in April and is charged with conspiracy to commit kidnapping, robbery, murder, kidnapping, and defeating the ends of justice and has been receiving treatment at Valkenberg psychiatric hospital his arrival in South Africa.
Before being put on a plane to South Africa, he fought his extradition for more than three years and was admitted to hospital in Britain for depression and post-traumatic stress disorder.
Prosecutors argue that Dewani conspired with Cape Town residents Zola Tongo, Mziwamadoda Qwabe and Xolile Mngeni to kill his wife two weeks after their lavish wedding.
Taxi driver Tongo, Qwabe and Mngeni are already serving prison sentences in connection with the murder.
The trial is expected to last for around two months, presided over by judge Jeanette Traverso.
Before his extradition, Dewani was detained in a hospital in Britain for depression and post-traumatic stress disorder.
Dewani is charged with conspiracy to commit kidnapping, robbery with aggravating circumstances, murder, kidnapping, and defeating the ends of justice.
Anni died when she was shot in the neck as the couple travelled in a taxi on the outskirts of Cape Town.
Dewani claimed he and his wife were kidnapped at gunpoint as they drove through Gugulethu in Cape Town.
He was released unharmed, but his wife's body was found in the abandoned car the next day.