Valtrate is the major active component of iridoids in Valeriana jatamansi plants, and has been widely used for the treatment of various diseases, giving rise to rapidly increasing market demands. The gene VjG10H from V. jatamansi encoding geraniol 10-hydroxylase (G10H) was introduced into V. jatamansi hairy roots and a transgenic hairy root culture system of V. jatamansi with good valtrate production ability was successfully established for the first time. The valtrate content of VjG10H-transformed lines ranged from 8.12 to 10.77 mg/g, with an average valtrate content (9.52 mg/g) of all the four G lines being higher than the non-transgenic hairy root line. Overexpression of VjG10H significantly improved valtrate production, indicating that G10H played an important role in stimulating valtrate accumulation. MeJA treatment stimulated valtrate accumulation in all of the VjG10H overexpression recombinant lines compared to untreated cultures, indicating that methyl jasmonate treatment was another effective regulation target for metabolic engineering of valtrate biosynthesis in V. jatamansi. Thus, transgenic hairy root technology coupled with elicitor treatments may be a promising strategy to increase valtrate yield in V. jatamansi.