The analgesic effect and its mechanism of electroacupuncture (EA) on inflammatory pain, especially in the rat model of collagen-induced arthritis (CIA), have not yet been studied. This study was designed to investigate the analgesic effect and its cholinergic and serotonergic mechanism of EA in the CIA rat model. To induce CIA, male Sprague–Dawley rats were immunized with bovine type II collagen emulsified in Freund's incomplete adjuvant, followed by a booster injection 14 days later. The analgesic effect was evaluated by tail flick latency (TFL). After induction of arthritis, the inflammatory pain threshold decreased as time passed and there was no big change of the pain threshold after 3 weeks. Three weeks after the first immunization, low frequency EA stimulation (2 Hz, 0.07 mA, 0.3 ms) delivered to Zusanli (ST 36) for 30 min showed the analgesic effect. Also, the analgesic effect of EA was blocked by pretreatment with atropine (muscarinic cholinergic receptor antagonist, 1 mg/kg i.p.), spiroxatrine (5-HT1a receptor antagonist, 1 mg/kg i.p.), and ondansetron (5-HT3 receptor antagonist, 0.5 mg/kg i.p.), but not by pretreatment with ketanserin (5-HT2 receptor antagonist, 1 mg/kg i.p.). These results suggest that low frequency EA can relieve inflammatory pain in CIA and the analgesic effect of EA can be mediated by muscarinic cholinergic receptor, 5-HT1a and 5-HT3 receptors, but not by 5-HT2 receptor.