ETHNOPHARMACOLOGICAL RELEVANCE: The root of Valeriana fauriei Briq. (VF) is a folk medicine registered in the Japanese Pharmacopeia 18th Edition. Valeriana plants have been utilized as sedatives and antidepressants in Europe, but only a few pharmacological reports have focused on VF. AIM OF THE STUDY: This study aimed to determine the active ingredients of VF and the mechanisms underlying its antidepressant effects. MATERIALS AND METHODS: We investigated the binding activity of VF ethanol extract on rat serotonin transporter (SERT) by competing against (S)-citalopram with a 96-well plate assay system and high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Additionally, we evaluated the inhibitory effect of the VF extract and its constituents on [ RESULTS: VF ethanol extract significantly inhibited both (S)-citalopram binding to SERT and serotonin uptake into HEK293 cells in concentration-dependent manners. Several fractions of the VF extract demonstrated synergistic action for serotonin uptake inhibition. Kanokonyl acetate (KNA) inhibited serotonin uptake in a concentration-dependent manner, and this activity was significantly enhanced by adding kessyl glycol diacetate (KGD), the major component of VF, which is not as active on its own. In addition, we found a new compound, kanokol, which significantly enhanced the interaction of KNA and KGD. The activity due to the interaction accounted for approximately 40% of the total VF activity. We obtained the regression equations that predicted VF extract activity by KNA and KGD contents in the presence of kanokol. CONCLUSIONS: The antidepressant effects of VF via serotonin uptake inhibition are derived from the interactions among its constituents, KNA and KGD, which play crucial roles exhibiting the interaction. Additionally, a key compound that improves the interaction is the new compound kanokol.