OBJECTIVE In this study, we examined the effects of lactose on long-term high-fat-diet-induced obesity in rats. METHODS A total of 112 Sprague-Dawley strain female rats (6 weeks old) were divided into four groups: a basic control diet group (Cont), 10% lactose diet group (Lac), high-fat diet group (Fat), and high-fat with 10% lactose diet group (Fat+Lac). After 0, 7, 14, and 84 days from starting the experimental diet, the animals were fasted overnight and killed by bleeding from the abdominal aorta under anesthesia (n = 8 or 9/group). RESULTS After 84 days, the addition of lactose to the high-fat diet decreased the final body weight, body weight gain, fat accumulation, and the levels of serum leptin, serum triglycerides, and serum glucose significantly (p < 0.05). Although there was no significant difference in the levels of serum calcium and phosphorus between the Fat and Fat+Lac groups, lumbar vertebral bone mineral density was significantly higher in the Fat+Lac group than in the Cont group on Day 82. Interestingly, the level of serum 1alpha, 25-dihydroxyvitamin D(3) in the Fat+Lac group on Day 84 was reduced by 74% compared with the Fat group (p < 0.01), while there was no significant difference in serum parathyroid hormone levels between the Fat and Fat+Lac groups. CONCLUSIONS This is the first study to suggest that the addition of lactose to a long-term high-fat diet may regulate not only calcium metabolism but also fat deposition. Further studies on the mechanism of dietary lactose in the regulation of adiposity would provide valuable data for the prevention of long-term high-fat-diet-induced obesity.