Background: Low concentrations of circulating vitamin D are common with obesity and may represent a potential mechanism explaining the elevated risk of certain cancers and cardiovascular outcomes observed in individuals who are overweight or obese. Objective: The objective of this study was to investigate the effects of 12 mo of weight loss through caloric restriction, exercise intervention, or both on serum 25-hydroxyvitamin D [25(OH)D] concentrations. Design: Overweight and obese postmenopausal women (n = 439) were randomly assigned to 1 of 4 groups: 1) diet modification (n = 118), 2) exercise (n = 117), 3) diet + exercise (n = 117), or 4) control (n = 87). The diet intervention was a group-based reduced-calorie program with a 10% weight-loss goal. The exercise intervention consisted of 45 min of moderate-to-vigorous intensity aerobic activity daily for 5 d/wk. Serum 25(OH)D concentrations were measured by using a competitive chemiluminescent immunoassay at baseline and 12 mo. Results: No significant change in serum 25(OH)D was found between the intervention and control groups. Women who lost <5%, 5-9.9%, 10-14.9%, or ≥15% of baseline weight had mean increases in 25(OH)D of 2.1, 2.7, 3.3, and 7.7 ng/mL, respectively (P for trend = 0.002). Baseline vitamin D status did not modify the effect of the interventions on weight loss or body-composition changes at the 12-mo follow-up. Conclusion: A greater degree of weight loss, achieved through either a reduced-calorie diet or increased exercise, is associated with increased circulating 25(OH)D concentrations. This trial is registered at clinicaltrials.gov as NCT00470119.