Objectives: Pharmacopuncture which is a combination of acupuncture and herbal medicine helps to prevent and treat the diseases and symptoms including various pains. However, little was known about the therapeutic effects and its mechanisms on acute pain, although pharmacopuncture has been used frequently in acupuncture clinics. Acupuncture is known for producing analgesia for persistent ankle sprain pain in human. Furthermore, it also produces analgesia in a rat model of ankle sprain pain. Methods: To illuminate the underlying mechanisms of capsaicin pharmacopuncture-induced analgesia, weight bearing force (WBF) was observed on the acute ankle sprained rat model. Ankle sprain was induced in the rat by manually hyper-extending ligaments of the right ankle. Capsaicin pharmacopuncture was applied to SI6 (Yanglo) on the left forelimb (contralateral to the sprained ankle). Results: In behavioral test, capsaicin pharmacopuncture produced marked analgesic effects on acute ankle sprained animals as measured by WBF of the affected limb similar to manual acupuncture. Capsaicin pharmacopuncture was also suppressed by serotonin (5-HT) receptor antagonist methysergide (2 mg/kg, Lp.), but not by opioids receptor antagonist naltrexone (10 mg/kg, Lp.) and alpha adrenoceptor antagonist phentolamine (5 mg/kg, Lp.). Conclusion: The data suggest that capsaicin pharmacopuncture-induced analgesia is accomplished by activating the descending serotonergic inhibitory systems.