Opioid dependence is an increasing clinical and public health problem. Current pharmacotherapies have limited efficacy and cause serious side effects. Increasing bodies of evidences suggest the neuropeptide, oxytocin (OT), as a potential treatment for drug abuse disorders. The current study was designed to evaluate the effect of OT on withdrawal, craving and anxiety scores, cortisol and dehydroepiandrosterone sulphate (DHEAS) blood level in heroin-dependent male patients. This randomized, double-blind placebo-controlled clinical trial was conducted on 58 males with opioid dependence by Abstinence Center of Addictive People in Iran. The participants were randomly allocated to receive intranasal OT (single dose; 40 IU, n = 29) or placebo (n = 29). Heroine withdrawal, craving and anxiety scores were measured using the Opioid Withdrawal Scale, Visual Analogue Scale and (Desire for Drug Questionnaire), and Hamilton checklist respectively. The cortisol and DHEAS levels at baseline and different post-intervention time were measured using a competitive immunoanalysis method. Acute OT administration reduced craving and withdrawal scores but did not change anxiety significantly. Single dose of OT decreased the level of cortisol and improved the cortisol/DHEAS ratio in the heroin users during abstinence (p < 0.01). These results suggest that OT may be useful in the attenuation of craving, withdrawal symptom in heroin-dependent patients and might be considered a new potential treatment for heroin dependence where positive effects of OT on stress-related hormones may be involved in this effect of OT.