Previous studies have shown that the sigma-1 receptor chaperone (Sig-1R) antagonists enhance the antinociceptive effects of opioid receptor agonists, and its selective agonist SA4503 attenuates the rewarding effects of morphine. These findings suggest that Sig-1R regulates μ-opioid receptor (MOR) signaling cascade. However, a little information regarding the involvement of Sig-1R in physical dependence induced by morphine is available. Therefore, the present study was designed to investigate the involvement of Sig-1R in physical dependence on morphine in mice. Morphine was injected by slow release emulsion, and withdrawal signs were precipitated by the injection of naloxone. Withdrawal signs precipitated by naloxone in morphine-dependent mice, especially body-weight loss and diarrhea, were almost completely suppressed by the Sig-1R antagonist NE100. Furthermore, we also found that protein levels of Sig-1R were potently increased in the proximal colon, which is taking part in absorption of water. These findings suggest that Sig-1R plays an important role in peripheral withdrawal signs in morphine-dependent mice. On the other hand, previous results showed that Sig-1R could physically associate with MOR. We hypothesized that the change of association between these receptors is involved with physical dependence. Therefore, the present study was also designed to investigate the association between FLAG-tagged Sig-1R and HA-tagged MOR in CHO cells. Sig-1R and MOR could form a complex in CHO cells as a co-immunoprecipitant and the association between Sig-1R and MOR was increased by treatment with morphine. It should be noted that co-immunoprecipitated MOR with Sig-1R was deglycocylated. These results suggest that Sig-1R regulates MOR signal transduction by deglycosylating MOR by forming a complex in CHO cells. In conclusion, the present study demonstrated that Sig-1R plays an important role in the expression of withdrawal signs in the morphine-dependent state, and Sig-1R may regulate the MOR-mediated signal transduction by forming a MOR-Sig-1R complex.