ABSTRACT Background Although there are many evidence-based clinical studies examining the effect of motivational interviewing (MI) on substance use rates and relapse in substance use disorder (SUD), it has not been adequately explained how motivational interviewing affects the nature of the disease. Objective This study was conducted to examine the effect of MI on treatment motivation and psychological well-being of patients with SUD. Methods This was a randomized controlled study, including 74 patients who were diagnosed with SUD. Patients were allocated either to a combination of MI and treatment as usual (intervention group, n = 38), or treatment as usual only (control group, n = 36). Personal Information Form, Treatment Motivation Questionnaire (TMQ), and the Psychological Well-Being Scale (PWB) were used to collect the data. Results TMQ subscales scores and PWB scores were significantly higher in the intervention group when compared with the control group after three weeks of treatment. Conclusions Our results showed that MI interventions significantly increase psychological well-being and treatment motivation in patients with SUD (p < .05). MI interventions may be used along with other treatments by psychiatric nurses and other mental health professionals in patients with SUD.