There is an unmet need for appealing and functional barley β-glucan (BG) food matrices that can provide sufficient and active BG doses to consumers. We investigated how molecular mass and oligomer structure important for BG food and health properties affected plasma lipids and gut parameters in hypercholesterolemic rats. Following 3 weeks on a high-cholestrol diet, rats were given a high-cholesterol diet supplemented with either cellulose (control) or purified barley BGs with low (100 or 150 kDa; glucagel or lowBG, respectively) or medium (530 kDa; mediumBG) molecular masses varying in cellotriosyl/cellotetraosyl oligomer ratio for 4 weeks. All four diets (control, glucagel, lowBG or mediumBG) reduced plasma triacylglycerol and cholesterols from week 3 to 7. The BG diets increased cecal production of short-chain fatty acids (SCFAs) compared to the control diet. The glucagel and lowBG diets stimulated the number of Bifidobacterium in the cecum, whereas the mediumBG diet reduced numbers of both Bacteroides/Prevotella and Lactobacillus in the cecum compared to the control diet. In conclusion, barley BGs at 6.5–7.5% of the diet independent of molecular mass and oligomer block structure showed no additional effect compared to the control treatment on blood cholesterol and triacylglycerol levels in this hypercholesterolemic rat model. Furthermore, the cecal fermentation pattern and microbial composition did not seem to affect plasma lipid composition.