BACKGROUND Plant sterols combined with exercise improves lipid profiles, however, the mechanism by which this lifestyle therapy alters whole body cholesterol metabolism remains unclear. OBJECTIVE This study examined the combined and individual effects of plant sterols and exercise on cholesterol absorption and synthesis in hypercholesterolemic adults. METHODS In an 8-week, parallel-arm trial, 84 subjects were randomized to 1 of 4 groups: plant sterols and exercise, plant sterols, exercise, or control. Cholesterol absorption and synthesis were measured using the single isotope single tracer technique, and deuterium incorporation approach, respectively. RESULTS Total cholesterol and triglyceride levels decreased (P < 0.01) by 7.7% and 11.8%, respectively, while HDL cholesterol levels increased (P < 0.01) by 7.5% in the combination group. Cholesterol absorption was 16% lower (P < 0.01) in the combination group, and 18% lower (P < 0.01) in plant sterol group, relative to control. Exercise had no effect on cholesterol absorption. Non-significant increases in cholesterol synthesis rates of 63% (0.084 ± 0.014 pools/d), 59% (0.075 ± 0.013 pools/d), 57% (0.072 ± 0.011 pools/d), were observed in the combination, exercise, and plant sterol groups, respectively, relative to control (0.031 ± 0.019 pools/d). LDL cholesterol levels correlated with cholesterol absorption, as represented by the area under the enrichment curve (r = 0.23, P=0.05), and with percent absorption relative to control (r = 0.25, P=0.03). CONCLUSION The combination of plant sterols and exercise improves lipid profiles primarily by suppressing cholesterol absorption.