β-Glucan, a soluble fiber with viscous property, has a documented cholesterol-lowering effect. The molecular weight (MW) of β-glucan, which contributes to viscosity, and an individual's genotype might influence the cholesterol-lowering efficacy of β-glucan.This study was designed to determine whether the cholesterol-lowering efficacy of barley β-glucan varied as a function of MW and the daily dose consumed. Our second aim was to determine whether any gene-diet interactions are associated with the cholesterol-lowering efficacy of β-glucan.In a randomized controlled crossover trial, 30 mildly hypercholesterolemic adults [12 men and 18 women, aged 27-78 y; body mass index (in kg/m(2)): 20-40; total cholesterol (TC): 5.0-8.0 mmol/L; LDL cholesterol: 2.7-5.0 mmol/L] were randomly assigned to receive a breakfast that contained either barley β-glucan at 3 g high MW (HMW)/d, 5 g low MW (LMW)/d, or 3 g LMW/d or a control diet, each for 5 wk. The washout period between the phases was 4 wk. Fasting blood samples were collected at the start and end of each phase for blood lipid analysis and genotyping.Consumption of 3 g HMW β-glucan/d lowered TC by -0.12 mmol/L (95% CI: -0.24, -0.006 mmol/L) compared with the control diet (P= 0.0046), but the LMW β-glucan, at either 3 g/d or 5 g/d, did not change serum cholesterol concentrations. This effect of HMW β-glucan was associated with gene-diet interaction, whereby individuals with the single nucleotide polymorphism (SNP) rs3808607-G allele (GG or GT) of the cytochrome P450 family 7 subfamily A member 1 gene (CYP7A1) had greater responses to 3 g HMW β-glucan/d in lowering TC than TT carriers (P= 0.0006).The HMW β-glucan rather than LMW β-glucan reduced circulating TC effectively in mildly hypercholesterolemic adults. The cholesterol-lowering effect of β-glucan may also be determined by the genetic characteristics of an individual. These data show that individuals carrying theCYP7A1SNP rs3808607-G allele are more responsive to the cholesterol-lowering effect of β-glucan with HMW than TT carriers. This trial was registered atclinicaltrials.govasNCT01408719.