Objectives: This study aimed at investigating the pharmacological and physiological effects of cannabidiol (CBD) oil on weight loss in diet-induced obese (DIO) mice. Methods: A DIO mice model was constructed with 33 C57BL/6 male mice, aged six weeks, who had been fed a high-fat diet for 13 weeks. Subsequently, 20 mg/kg (n=11) or 60 mg/kg (n=11) of CBD oil or sesame seed oil (n=11) per day was given along with a high-fat diet for four weeks. The body weight of each subject was measured weekly, and venous blood was drawn for biochemistry and enzyme-linked immunoassay before and after the four-week trial period. An oral glucose tolerance test was performed to assess glucose metabolism. At the end of the CBD oil treatment, dual-energy X-ray absorptiometry was used to calculate body fat composition, and the mesenteric adipose tissue was measured as representative of the fat mass of each subject. For statistical analysis, we used the Kruskal-Wallis test, Turkey’s test using ranks and generalized estimating equations. Results: After administration of CBD oil (60 mg/kg) for four weeks, the DIO mice showed significant weight loss, compared to the sham control mice (p=0.027). Mice fed with 60 mg/kg of CBD oil also had a significant reduction in fat percentage (p=0.009) and mesenteric fat weight loss (p=0.024), compared to the sham control mice, even with higher food intake (p=0.029). Moreover, mice fed with 60 mg/kg of CBD oil showed a significant improvement in glucose tolerance (p=0.003) and lower plasma leptin levels (p=0.006). Conclusion This study shows that orally administered CBD oil induces weight loss in DIO mice. It has been postulated that CBD oil attenuates an over-activated endocannabinoid system, thereby increasing energy expenditure, and improving glucose metabolism and leptin resistance.