Overweight is associated with both higher bone mineral density (BMD) and higher serum leptin concentrations. In humans, little is known about the relationship of leptin concentration and bone density. We studied this relationship in a large, national population-based sample. Participants included 5815 adults in the Third U.S. National Health and Nutrition Examination Survey (NHANES III; 1988–1994) who underwent DXA of the proximal femur and measurement of fasting serum leptin. Mean ± SE BMD (gm/cm2) of the total hip was 1.01 ± 0.005 in men, 0.94 ± 0.004 in premenopausal women, and 0.78 ± 0.007 in postmenopausal women. Bone density increased with increasing leptin concentration in men (p = 0.003), premenopausal women (p < 0.001), and postmenopausal women (p < 0.001). However, after adjusting for body mass index (BMI) and other bone density-related factors, an inverse association emerged in men (p < 0.001), being most evident among men <60 years old. There was no association of leptin and BMD in premenopausal women (p = 0.66) or postmenopausal women (p = 0.69) in multivariate analysis. Controlling for leptin had no effect on the strong positive association of BMI and BMD in either men or women. Serum leptin concentration did not appear to affect directly BMD. If present, the association appeared to be limited to younger men who are at lower risk of osteoporosis.