Lupin kernel fiber beneficially modifies blood lipids because of its bile acid-binding capacity. The aim of this study was to evaluate the preventive effects of a lupin kernel fiber preparation on cardiovascular diseases and to clarify possible mechanisms. In a randomized, double-blind, controlled crossover trial, 60 moderately hypercholesterolemic adults (plasma total cholesterol: >5.2 mmol/L) passed 3 intervention periods in different orders with a 2-wk washout phase between each. Participants consumed either a high-fiber diet containing 25-g/d lupin kernel fiber (LF) or citrus fiber (CF), or a low-fiber control diet (CD) for 4 wk each. Anthropometric, plasma, and fecal variables were assessed at baseline and after the interventions. Contrary to the CF period, total (9%) and LDL (12%) cholesterol as well as triacylglycerols (10%) were lower after the LF period when compared with the CD period [P ≤ 0.02, adjusted for baseline, age, gender, and body mass index (BMI)]. HDL cholesterol remained unchanged. Moreover, the LF period reduced high-sensitivity C-reactive protein (P = 0.02) and systolic blood pressure (P = 0.01) when compared with baseline. Bile acid binding could not be shown because the excretion of total bile acids remained constant after the high-fiber diets. However, the LF period resulted in an enhanced formation of the main short-chain fatty acids in comparison with the CD period. During the CF period, only acetate increased significantly. Both high-fiber diets led to higher satiety and modified nutritional behavior, resulting in significantly lower body weight, BMI, and waist circumference compared with the CD period. The blood lipid-lowering effects of LF are apparently not a result of bile acid binding. Rather, we hypothesize for the first time, to our knowledge, that the blood lipid-lowering effects of LF may be mainly attributed to the formation of short-chain fatty acids, specifically propionate and acetate. This trial was registered at clinicaltrials.gov as NCT01035086.