The prosecutor said Walter James Palmer, a dentist from Minnesota, has also been accused by wildlife officials of killing the animal without a permit on July 1.
He said Palmer, who has left Zimbabwe, says he killed the lion but believed it was a legal hunt.
He said the local hunter, Theo Bronkhorst, and private game park owner Honest Ndlovu, who assisted Palmer, were escorted into the courthouse in Hwange west of Harare, by plain-clothes detectives.
The prosecutor said Palmer, on Tuesday, said he had hired professional guides who secured hunting permits and deeply regretted taking the lion.
He added he had not been contacted by authorities in Zimbabwe or the U.S. and would assist in any inquiries.
The two Zimbabweans could be fined 20,000 dollars and possibly jailed for up to 10 years, if found guilty.
The university’s Wildlife Conservation Research Unit said Cecil was fitted with a GPS collar for a research project by scientists from Oxford University and was one of the oldest and most famous in Zimbabwe.
It said it had been tracking Cecil since 2008 and was “deeply saddened” by his death.
“Insofar as this happened illegally, we consider it deeply reprehensible. We are working closely with Zimbabwe’s National Parks authorities to support their “meticulous work” in prosecuting the case’’, it said.
The unit also said Cecil’s death would likely trigger a power struggle in the pride, resulting in the death of other male lions as well as Cecil’s offspring.
“When a male lion is killed, because of the way their society works, a likely consequence is the overthrow and death of other adult male members of his weakened coalition, and the subsequent infanticide of his cubs.
Johnny Rodrigues, Zimbabwe Conservation Task Force Chairman, said Cecil was lured out of the park with bait before being shot.
The incident has triggered fierce debate over the commercial ‘trophy’ hunting of African big game.
Edward Bourke, Chairman of the Australia-based Saving the Lion Foundation, said Cecil’s death showed the dangers of legal hunting.
“There is enough global pressure to push for change. There is an opportunity to offer alternatives; including international aid for establishing safe haven environments like national parks or eco-tourism zones”, he said.
According to a report, Palmer’s hunting has attracted scrutiny in the past.
It said he pleaded guilty to lying to a U.S. wildlife agent in 2008 about a black bear he killed in Wisconsin two years before.
It said he was accused of killing it 40 miles outside a permitted zone, hauling the carcass back into the approved area and certifying falsely that it was killed there.
He was sentenced to one year probation and fined 2,938 dollars.