Objective: Barley fiber rich in beta-glucans lowers serum lipids, but is difficult to incorporate into products acceptable to consumers. We investigated the physiological effects of two concentrated barley β-glucans on cardiovascular disease (CVD) endpoints and body weight in human subjects. Methods: Hypercholesterolemic men and women (n = 90) were randomly assigned to one of two treatments: low molecular weight (low-MW) or high molecular weight (high-MW) concentrated barley β-glucan consumed as a daily supplement containing 6 grams beta-glucan/day. Fasting blood samples were collected at baseline and week 6 and analyzed for total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, glucose, insulin, homocysteine and C-reactive protein (CRP). Dietary intakes, body weights, blood pressure, hunger ratings, and gastrointestinal symptoms were measured at baseline and 6 weeks. Results: The only difference between treatments in lipid outcomes at week 6 was a reduction of the cholesterol/HDL ratio in the low-MW group and a small increase in the high-MW group. No changes were found in blood pressure, glucose, insulin, and gastrointestinal symptoms. Body weight decreased from baseline to 6 weeks in the high-MW group while body weight increased in the low-MW group. Levels of hunger decreased slightly in the low-MW group and decreased significantly in the high-MW group (P = 0.02) Conclusion: Overall, supplementation with isolated barley β-glucans of different molecular weights had small effects on cardiovascular disease markers. Molecular weight of the barley fiber did alter effects on body weight with the high-MW fiber significantly decreasing body weight.