Purpose. This study aimed to investigate the analgesic effect and mechanism of electroacupuncture (EA) in nicotine withdrawal-induced hyperalgesia rats. Methods. Behavioral testing was conducted twice a week for 7 weeks during nicotine administration using von Frey filaments. Electroacupuncture at the bilateral “Zusanli” and “Taichong” points was applied daily for 3 days during nicotine withdrawal. Western blot analysis and immunohistology were used to determine expression levels of pain-related factors in the spinal cord and midbrain periaqueductal gray (PAG). Results. Behavioral tests showed that electroacupuncture had a significant analgesic effect on nicotine withdrawal-induced hyperalgesic rats. Western blot results demonstrated that, in hyperalgesic rats, the expressions of nicotinic acetylcholine receptors (subunits: nAChR α7, α4, or β2) decreased in the spinal cord, nAChR α7, and β2 decreased in PAG. The proinflammatory factor cyclooxygenase 2 (COX2) and the activated microglia (ionized calcium-binding adaptor molecule 1, Iba1 positive cells) increased in the spinal cord and PAG compared to controls. After electroacupuncture treatment, nAChR α7 and nAChR β2 expressions increased significantly, and COX2 and Iba1 expressions decreased in the spinal cord. Compared with the nonelectroacupuncture nicotine withdrawal group, electroacupuncture stimulation increased the expression of nAChR α7 and nAChR α4 in the PAG of rats with electroacupuncture. Immunohistochemical results confirmed that electroacupuncture reversed nicotine withdrawal-induced changes in nAChR α7 positive neurons and Iba1-positive microglia in the dorsal horn of the spinal cord. Conclusion. Electroacupuncture treatment has an analgesic effect on nicotine withdrawal-induced pain in nicotine-dependent rats. The mechanism of analgesia of the electroacupuncture treatment relates to the increased expression of nAChR α7 and nAChR β2 proteins in the spinal cord, nAChR α7 in the PAG, and decreased expression of Iba1 and COX2 protein in the spinal cord.