Consumers’ demand for functional foods of plant origin that would positively affect their health and wellbeing is successively increasing. This affords possibilities for reducing the volume of waste from the brewing industry through the exploitation of the nutritional and health-protective potential of the brewer’s spent grain. Inclusion of brewer’s spent grain to food products contributes to increased contents of dietary fiber and protein in diet, to increased fecal bulk, to accelerated excretion of fat and cholesterol as well as to accelerated peristalsis. The health-promoting properties of dietary fiber and beta-glucans have been confirmed in ample studies which demonstrated their hypocholesterolemic and hypoglycemic effects that are implied to reduce the risk of development of diet-related diseases like dyslipidemia, cardiovascular diseases, obesity, and type 2 diabetes. The objective of this study was to evaluate the effect of the addition of brewer’s spent grain to high fat diets with canola oil on selected biochemical markers in blood of Wistar rats. The study demonstrated a positive effect of the addition of a high-fiber preparation from brewer’s spent grain to a high fat diet with lard for rats on reduced concentrations of LDL-cholesterol fraction and triglycerides, and on increased concentration of HDL-cholesterol fraction their blood serum.