Abstract Background and aim: Removal of the testes (gonadectomy; GDX), the primary source of androgens, increases anxiety behavior in several tasks. Opioids are known to play a role in mediating the effects of androgen. In the present study, the effect of testosterone and opioidergic system on anxiety behavior was investigated. Methods Adult male Wistar rats were bilaterally castrated. The elevated plus maze which is a useful test to investigate the effects of anxiogenic or anxiolytic drugs in rodents was used. Results The data indicated that there is a decrease, 10 days after castration, in the percentage of OAT (the ratio of time spent in the open arms to total times spent in any arms×100) and OAE (the ratio of entries into open arms to total entries×100) but not locomotor activity, showing anxiogenic-like effects of gonadectomy. Intraperitoneal injection of testosterone (200, 300 and 450mg/kg) and morphine (2.5, 5 and 7.5mg/kg), before testing 10 days after castration, showed an increase in OAT and OAE. Furthermore, injection of naloxone (5 and 7.5mg/kg, i.p.), 5min before testing 10 days after castration, decreased OAT and OAE. Also, injection of a significant dose of testosterone (300mg/kg, i.p.), 1h before the injection of different doses of morphine (1, 2.5, 5 and 7.5mg/kg, i.p.), 10 days after castration, did not significantly alter OAT, OAE and locomotor activity. While, administration of a significant dose of testosterone (300mg/kg, i.p.), 1h before the infusion of different doses of naloxone (1, 2.5, 5 and 7.5mg/kg, i.p.), 10 days after castration, decreased OAT and OAE. Conclusion The results show the involvement of testosterone and opioidergic system in anxiogenic-like behaviors induced by gonadectomy.