We previously reported that nicotine (NIC)-induced analgesia was elicited in part by activation of the endogenous opioid system. Moreover, it is well known that NIC has physical-dependence liability, but its mechanism is unclear. Therefore, we examined whether physical dependence on NIC was mediated by activation of the endogenous opioid system in ICR mice. We evaluated increased serum corticosterone (SCS) as an indicator of NIC withdrawal, as it is a quantitative indicator of naloxone (opioid receptor antagonist, NLX)-precipitated morphine withdrawal in mice. In this study, NLX precipitated an SCS increase in mice receiving repeated NIC, by a dose-dependent mechanism, and correlated with the dose and number of days of repeated NIC administration. When an opioid receptor antagonist (naltrexone) was concomitantly administered with repeated NIC, the NLX-precipitated SCS increase was not elicited. Concomitant administration of the α7 nicotinic acetylcholine receptor (nAChR) antagonist (methyllycaconitine) with repeated NIC, but not the α4β2 nAChR antagonist (dihydro-β-erythroidine), did not elicit an SCS increase by NLX. Thus, a physical dependence on NIC was in part mediated by the activation of the endogenous opioid system, located downstream of α7 nAChR.