The purpose of this study was to determine the independent and combined effects of plant stanol ester (PSE) margarine and aerobic exercise on blood lipid concentrations and related intravascular enzymes in 26 healthy sedentary, middle-aged men and postmenopausal women (age, 53 ± 8 years; body mass index, 27 ± 1.0, % fat, 28.5 ± 2). In a stratified double-blind manner, participants were randomly assigned to either a PSE (n = 17) or a placebo (CON, n = 9) margarine group. Participants supplemented their daily diets with 42 g of margarine spread (PSE = 3 g; CON, PSE = 0 g, of approximately equal energy content) for 9 weeks. During the last 4 weeks of margarine supplementation (MS), participants expended 400 kcal on a treadmill 5 d/wk at 65% of V̇o2 reserve (2000 kcal/wk). Fasting blood samples were obtained before initiating and after 4 weeks of MS and after exercise training. All blood samples were analyzed for total cholesterol, low-density lipoprotein cholesterol, triglyceride, high-density lipoprotein cholesterol (HDL-C), hepatic lipase, lipoprotein lipase, and cholesterol ester transfer protein activities. Total cholesterol (-10%), low-density lipoprotein cholesterol (-13%), and triglyceride (-18%) concentrations decreased after 4 weeks of MS in the PSE group, but not in the CON group (P < .05 for all). Four weeks of aerobic exercise increased HDL-C by 21% in the CON group (P < .05) and by 4% in the PSE group (P > .05). Total cholesterol-HDL-C ratio decreased significantly (P < .05) in the PSE group, but not in the CON group. No other significant alterations were observed with either PSE or exercise. Our findings suggest that PSE is effective in reducing blood cholesterol concentrations and that exercise can increase HDL-C in middle-aged men and postmenopausal women. Our findings also suggest that PSE supplementation may attenuate the exercise-induced increase in HDL-C. © 2006 Elsevier Inc. All rights reserved.