The roots and rhizomes of Valeriana jatamansi have long been used as folk medicinein Asia and usually named as “Zhizhuxiang” in Chinese for the treatment of abdominaldistention and pain. However, its active ingredients and molecular targets for treatmentof abdominal pain remain unrevealed. Inhibitors of Ca v 2.2 N-type voltage-gated calciumchannels (VGCCs) are actively sought after for their potential in treating pain, especiallychronic pain. As far as we know, the method used for seeking analgesic activeingredient from plant material has rarely been reported. The analgesic potentials ofthe EtOH extract (0.01 mg/ml) of the roots and rhizomes of V. jatamansi and itsEtOAc, n-BuOH and H 2 O soluble parts (0.01 mg/ml, respectively) were tested hereinon Ca v 2.2, using whole-oocyte recordings in vitro by tow-electrode voltage clamp.The results indicated that the EtOAc-soluble part exhibited the most potent inhibitionof Ca v 2.2 peak current (20 mv). The EtOAc-soluble part was then subjected to silicagel column chromatography (CC) and giving 9 fractions. Phytochemical studies werecarried out by repeated CC and extensive spectroscopic analyses after the fraction(0.01 mg/ml) was identified to be active and got seventeen compounds (1–17). Allisolates were then sent for further bioactive verification (1 and 3 at concentration of10 µM, others at 30 µM). In addition, the selectivity of the active compounds 1 and 3were tested on various ion channels including Ca v 1.2, Ca v 2.1 and Ca v 3.1 VGCCs andKv1.2, Kv2.1, Kv3.1 and BK potassium channels. The results indicated that compound1 and 3 (an abundant compound) inhibited Ca v 2.2 with an EC 50 of 3.3 and 4.8 µM,respectively, and had weaker or no effect on Ca v 1.2, Ca v 2.1 and Ca v 3.1 VGCCsand Kv1.2, Kv2.1, Kv3.1 and BK potassium channels. Compounds 1 and 3 appearto act as allosteric modulators rather than pore blockers of Ca v 2.2, which may playcrucial role in attenuating nociception. The results of present research indicated that theethnopharmacological utilization of V. jatamansi for relieving the abdominal distentionand pain may mediate through Ca v 2.2 channel. Our work is the first demonstrationof inhibition of Ca v 2.2 by iridoids, which may provide a fresh source for finding newanalgesics.