Bone marrow fat is a unique fat depot that may regulate bone metabolism. Marrow fat is increased in states of low bone mass, severe underweight, and diabetes. However, longitudinal effects of weight loss and improved glucose homeostasis on marrow fat are unclear, as is the relationship between marrow fat and bone mineral density (BMD) changes. We hypothesized that after Roux‐en‐Y gastric bypass (RYGB) surgery, marrow fat changes are associated with BMD loss. We enrolled 30 obese women, stratified by diabetes status. Before and 6 months after RYGB, we measured BMD by dual‐energy X‐ray absorptiometry (DXA) and quantitative computed tomography (QCT) and vertebral marrow fat content by magnetic resonance spectroscopy. At baseline, those with higher marrow fat had lower BMD. Postoperatively, total body fat declined dramatically in all participants. Effects of RYGB on marrow fat differed by diabetes status (p = 0.03). Nondiabetic women showed no significant mean change in marrow fat (+1.8%, 95% confidence interval [CI] –1.8% to +5.4%, p = 0.29), although those who lost more total body fat were more likely to have marrow fat increases (r = –0.70, p = 0.01). In contrast, diabetic women demonstrated a mean marrow fat change of –6.5% (95% CI –13.1% to 0%, p = 0.05). Overall, those with greater improvements in hemoglobin A1c had decreases in marrow fat (r = 0.50, p = 0.01). Increases in IGF‐1, a potential mediator of the marrow fat‐bone relationship, were associated with marrow fat declines (r = –0.40, p = 0.05). Spinal volumetric BMD decreased by 6.4% ± 5.9% (p < 0.01), and femoral neck areal BMD decreased by 4.3% ± 4.1% (p < 0.01). Marrow fat and BMD changes were negatively associated, such that those with marrow fat increases had more BMD loss at both spine (r = –0.58, p < 0.01) and femoral neck (r = –0.49, p = 0.01), independent of age and menopause. Our findings suggest that glucose metabolism and weight loss may influence marrow fat behavior, and marrow fat may be a determinant of bone metabolism. © 2017 American Society for Bone and Mineral Research.