Mirabilis himalaica is an endangered plant species that has been used as an herb by native Tibetan people because of its rotenoid composition. In the present study, the effects of different factors including explants, preculture time and bacterial infection time on the induction rate of hairy roots were investigated. The highest hairy root induction rates were obtained using top leaves as explants, a preculture time of 2 d for young leaves, and Agrobacterium infection of young leaves for 20 min. Genomic PCR confirmed that the rol genes were integrated into the genome of M. himalaica. Different liquid media including MS, 1/2 MS, B5 and 1/2 B5 had significantly different influences on biomass and rotenoid accumulation in hairy root cultures. The hairy root cultures grew better in 1/2 MS and 1/2 B5 than in MS and B5, while a higher level of rotenoid was obtained in hairy root cultures growing in MS and 1/2 MS liquid media. Further, the contents of rotenoid were detected by HPLC in wild roots (0.176 mg.g-1 DW), stems (0.168 mg.g-1 DW) and leaves (0.157 mg.g-1 DW) of M. himalaica. In particular, the hairy root cultures had the highest rotenoid content (0.755 mg.g-1 DW) in 1/2 MS liquid medium, which was 4.3 times higher than in wild type roots (0.176 mg.g-1 DW).