Objective: To determine how moderate weight loss protocol through diet and exercise may affect changes in body composition, to determine the effects of weight loss on cholesterol metabolism and to examine the relationship between cholesterol metabolism and changes in body composition. Design: Thirty-five otherwise healthy, hypercholesterolemic women completed a 24-week weight loss study. A 20% decrease in energy intake through diet and a 10% increase in energy expenditure by exercise were combined with motivational strategies to encourage weight loss. The diet was self-selected and comprised of 50-60% carbohydrates, 20% protein and o30% fat. Results: Participants lost an average of 11.772.5 kg (Po0.001). Whole body and regional losses in tissue mass occurred after weight loss. After weight loss, cholesterol fractional synthesis rate (FSR) decreased (P ¼ 0.003) 3.8679.33%, whereas rates of cholesterol absorption and turnover did not change (3.31719.4%, P ¼ 0.32 and À0.0176.20%, P ¼ 0.75, respectively). Changes in cholesterol turnover were positively correlated (r ¼ 0.44, P ¼ 0.01) with changes in FSR. Reductions in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) were predictive (b ¼ À5.04, r ¼ 0.38; P ¼ 0.03, and b ¼ À147, r ¼ 0.40; P ¼ 0.03, respectively) of increases in cholesterol turnover. Losses in skeletal muscle (SM) and upper-body SM predicted (b ¼ 6.82, r ¼ 0.36; P ¼ 0.04 and b ¼ 14.7, r ¼ 0.41; P ¼ 0.01, respectively) decreases in cholesterol absorption. Conclusions: Decreases in cholesterol synthesis after moderate weight loss are not compensated for by changes in cholesterol absorption or turnover. Changes in regional body composition were associated with variations in cholesterol metabolism. Understanding how weight loss affects cholesterol metabolism will help identify more effective treatment routes for overweight individuals undergoing weight loss resulting in earlier and more intensive therapy for the associated dyslipidemia.