A Zimbabwean man, Onward Muchangowa, and two South Africans were arrested in the neighbouring country after they were found in possession of two rhino horns.
Muchangowa and the two brothers, Deon and Niklaas van Deventer, who are accused of rhino poaching, appeared at the Makhado Magistrates' Court in Louis Trichardt on Monday and are expected back in court next week.
Media reports say the men are being charged with possession of two horns from a rhino that was poached at a Limpopo farm near Witpoort.
On their previous court appearance in June, magistrate Bennie Smit denied the brothers bail as he believed there was a likelihood for them to commit further offences.
Muchangowa was also denied bail after the court ruled he was a flight risk. The trio was arrested in January 2017 following a tip-off that they had attempted to sell rhino horns in the Makhado area.
The brothers were previously linked to a bloody trail of at least 22 dead rhinos which had been poached in various locations around South Africa in 2006.
In 2007, they were caught red-handed as they exited iMfolozi Game Park in Zululand.