Background: Pain is considered as a multidimensional conscious experience that includes a sensory component and a negative affective-motivational component. Electroacupuncture (EA) is widely used to treat pain and paininduced negative emotions, however, little is known about the mechanisms underlying the effect of EA. Objective: This study investigated the effect of EA on alleviating the anxiety-like behaviors in pain aversion rats and its anterior cingulate cortex (ACC) regulation mechanism. Methods: After a Freund’s complete adjuvant (CFA)-conditioned place aversion (C-CPA) model was established in rats, EA treatment (2/100 Hz, 30 min, once/day, 4 days totally) was applied at bilateral Zusanli (ST36) and Kunlun (BL60) acupoints. Von Frey filaments were used to measure changes of pain withdrawal threshold (PWT) at indicated time points. Elevated zero maze (EZM) was used to investigate the changes of pain-related anxiety and CPA was used to investigate the changes of pain aversion. The protein expression levels of GAD67, PV, and NPY in ACC were detected by Western blotting. Results: Compared with the control group, the staying time in the "CFA-paired compartment" was significantly reduced, and the PWT was decreased in model group. In the EZM assessment, the distance and the time in open arm, as well as the number of open arm entries of model group were significantly lower than those in the control group. In the CPA assessment, the time spent in the "CFA-paired compartment" was significantly decreased in model group compared with control group, and EA reversed the changes in pain sensation and in pain-related emotions. Western blotting showed that the NPY level, but not the levels of GAD67 and PV, was significantly increased in the ACC of the model group compared to that of the control group. The increased expression of NPY in the ACC was significantly downregulated by EA, while sham EA produced no such effect. Conclusion: EA can effectively relieve the pain and pain-related emotions, and its mechanism may be achieved by down-regulating the expression of NPY in the ACC.