Background: We evaluated the protective effects of melatonin against high-fat diet (HFD)-induced deterioration of bone microarchitecture using high-resolution peripheral quantitative computed tomography (HR-pQCT). Methods: Four-week-old male C57BL/6 mice were divided into control (chow diet group), HFD, and HFD + melatonin-administered groups. Mice were sacrificed after 14 weeks, and the right femur was extracted. The microskeletal structure of the femur was analyzed using SkyScan1173 (version 1.6). A 3-dimensional image was reconstructed using the Nrecon (version 1.7.0.4) program. Results: Bone volume (BV) was significantly increased in the HFD group compared with that in the normal diet group, and that of the melatonin group also increased significantly compared with BV of the normal diet group (p<0.05). Percent BV/total volume [TV] and bone surface/BV were significantly higher in both the HFD and melatonin groups than in the normal diet group (p<0.05), and the melatonin group had the highest BV/total volume (TV). BMD was lower in the HFD than in the normal diet group and was the highest in the melatonin group. Conclusions This study shows that melatonin inhibited the deterioration of microarchitecture induced by a HFD. A better understanding of the protective effect of melatonin on bone microarchitecture and mechanisms could provide fracture prevention for people who are obese.