In this study, the transformed system mediated by Agrobacterium tumefaciens of Gynostemma pentaphyllum was constructed by using the phosphomannose-isomerase (PMI) gene as a marker. To investigate the cefotaxime sodium salt (Cef) concentration of bacteriostatic medium and the appropriate mannose concentration in the selectable medium, explants of the stems with buds were cultured in a basic medium supplemented with different Cef and mannose concentrations, respectively. After these were optimized, 288 explants were transformed according the protocol described above to verify their availability by using the polymerase chain reaction (PCR), reverse transcription-PCR and chlorophenol red. The results showed that the appropriate Cef concentration for bacteriostatic culture and mannose concentration for selectable culture were 150 mg l⁻¹ and 3 g l⁻¹ for stem with buds, respectively. According to the PCR results, the transformation frequency of stems with buds was 20.49% with a regeneration rate of 29.16%. In future, the CPR assay could be the auxiliary method of choice as it is moderately accurate, but it has good maneuverability and is cost effective for large-scale use.