β-Glucans are known to exhibit hypocholesterolemic effects. Increased intestinal viscosity is thought to be crucial for cholesterol lowering. It is suggested that concentration, molecular mass, and structure, including the ratio of (1→3) to (1→4) glucan bonds in the molecule, are of importance for β-glucan functionality. This study investigated the effects of 3 different β-glucan sources, incorporated into a beverage and yogurt, on blood lipids and fecal endpoints. Fourteen participants completed this randomized, crossover, single-blinded study with four 3-wk periods: control and 3.3 g/d oat, barley, and barley mutant β-glucans of similar molecular mass. Before and after each period, fasting and postprandial blood samples were drawn and 3-d fecal samples were collected. Treatment did not affect changes in total, LDL, and HDL cholesterol compared with control; however, consumption of 3.3 g/d of oat β-glucans for 3 wk resulted in greater decreases in total (-0.29 ± 0.09 mmol/L, P < 0.01), LDL (-0.23 ± 0.07 mmol/L, P < 0.01), and HDL (-0.05 ± 0.03 mmol/L, P < 0.05) cholesterol compared with baseline. Changes in LDL in the β-glucan treatments were not related to β-glucan structure (cellotriosyl:cellotetraosyl). Decreases in fasting triacylglycerol were substantially greater after oat β-glucan treatment compared with control (P = 0.03). Fecal dry and wet weight, stool frequency, fecal pH, and energy excretion were unaffected. The results do not fully support the hypocholesterolemic effects by differently structured oat and barley β-glucans. However, substantial differences compared with baseline suggest a potential for oat β-glucan, presumably due to its higher solubility and viscosity. This underlines the importance of elusive structural β-glucan features for beneficial physiologic effects.