Clinically, acupuncture affects the motility of the extrahepatic biliary tract, but the underlining mechanisms are still unknown. We applied manual acupuncture (MA) and electrical acupuncture (EA) separately at acupoints Tianshu (ST25), Qimen (LR14), Yanglingquan (GB34), and Yidan (CO11) in forty guinea pigs (4 groups) with or without atropinization under anesthesia while Sphincter of Oddi (SO) myoelectric activities and gallbladder pressure were monitored. In both MA and EA groups, stimulation at ST25 or LR14 significantly increased the frequency and amplitude of SO myoelectrical activities and simultaneously decreased the gallbladder pressure as compared to the pre-MA and pre-EA (P < 0.05). On the contrary, stimulation at GB34 or CO11 significantly decreased SO myoelectricity and increased the gallbladder pressure (P < 0.05). Pretreatment with atropine could abolish the effect of stimulation at acupoints ST25, GB34 and LR14 (P > 0.05), although significant myoelectricity increases were still inducible with MA or EA stimulation at CO11 (P < 0.05). In summary, acupuncture has bi-directional effects to gallbladder pressure and SO function, which probably due to autonomic reflex and somatovisceral interactions.