An efficient transformation system for Ajuga multiflora Bunge was established by using Agrobacterium rhizogenes strain A4. After inoculation with the bacteria, we obtained a number of hairy-root clones from micro-calli of the explant petioles. One fast-growing line showed the highest production of 20-hydroxyecdysone (20-HE). PCR amplification of rooting locus (rol) genes revealed that the left hand-transferred DNA of the root-inducing plasmid was inserted into the genome of our transformed Ajuga hairy roots. This integration was further confirmed by DNA-DNA hybridization. The 20-HE content in hairy roots was 10 times higher than that measured in the wild type.