A simple and efficient protocol for Agrobacterium tumefaciens-mediated genetic transformation of Eupatorium adenophorum has been developed. Stem segments were pre-cultured on Murashige and Skoog (MS) medium for 24 h and then co-cultured with A. tumefaciens strain LBA4404 harboring the pBI121 plasmid on the same medium in darkness for 3 days. After co-cultivation, explants were transferred to MS medium supplemented with 1.0 mg l−1 naphthaleneacetic acid (NAA), 1.0 mg l−1 6-benzyladenine (BA), 150 mg l−1 kanamycin (Kan), and 200 mg l−1 cefotaxime (Cef), and incubated at 25 ± 1°C under a 16-h photoperiod. Callus formation was induced for 3–4 weeks. Induced calluses were transferred to fresh selection medium and subcultured every 15 days. After 6–8 weeks, regenerated plantlets from embryogenic callus could be observed. Buds of 1 cm in length were transferred to half-strength MS medium containing 150 mg l−1 Kan and 200 mg l−1 Cef, and rooted plantlets were obtained after 4 weeks. Putative transgenic plants were confirmed using GUS histochemical assay, polymerase chain reaction (PCR), and reverse transcriptase (RT)-PCR. A transformation efficiency of 2% was obtained.