Background: Recently we found the ethanolic extract and iridoid-rich fraction from Valeriana jatamansi Jones, which is a traditional Chinese medicine exhibited anxiolytic properties. Objective: This study aims to the substance dependence effect of the ethanolic extract and iridoid-rich fraction. Materials and Methods: The study included two experiments: Mice were given orally with ethanolic extract for 12 weeks or iridoid-rich fraction for 16 weeks in experiment I and experiment II, respectively. Diazepam was used as a control drug and the normal mice groups were administered with 0.5% carboxymethyl cellulose Na in both experiments. All groups were administered twice daily. Natural withdrawal symptoms, withdrawal-induced body weight change, audiogenic tail-erection test (in experiment I), and pentylenetetrazol (PTZ)-induced convulsion test (in experiment II) were measured. Results: (1) Compared to normal group in both experiments, the diazepam-treated group exhibited obvious withdrawal symptoms of tail-erection, irritability, teeth chattering, etc; the body weight of them after withdrawal had a period of significant loss (P < 0.05 or P < 0.01); and the ratios of tail-erection and seizure in two experiments were improved significantly when mice were induced by mixer noise ringtone (experiment I) or PTZ (experiment II) (P < 0.05 or P < 0.01).(2) In experiment I and II, there were no significant differences between mice that received ethanolic extract or iridoid-rich fraction and normal group in terms of natural withdrawal symptoms and withdrawal-induced body weight change (P > 0.05); in audiogenic tail-erection test, it found that the significant difference compared with normal group was just in ethanolic extract 900 mg/kg dose group on week 8 (P < 0.05); in PTZ-induced convulsion test, mice in iridoid-rich fraction groups had a slightly tail-erection and seizure, all results of them were with no significant difference compare to normal mice (P > 0.05), while significant lower than diazepam group (P < 0.01). Conclusion: (1) The two experiments successfully established the physical dependence of diazepam by gradually increasing the dose.(2)There were just a few mice received with ethanolic extract for 12 weeks or iridoid-rich fraction for 16 weeks appearing some slight withdrawal symptoms after precipitated withdrawal, but it didn't show significant difference compared to normal mice. Therefore, these indicated that the risks of potential drug dependence about ethanolic extract and iridoid-rich fraction were far lower than that of diazepam.