This study evaluated the effects of carnosine supplementation on carnosine concentration in muscles and blood biochemical indices of rats. Thirty-two eight-week-old Sprague-Dawley male rats were randomly divided into a control group (CON) as well as three carnosine-treated groups. The carnosine-treated groups included groups fed diets composed of 0.01% carnosine (LC), 0.1% carnosine (MC), and 1.0% carnosine (HC). Body weight gain, food intake, feed efficacy rate, protein efficacy rate, and organ weights were not significantly different among the groups. In all groups, the mean carnosine levels in gastrocnemius muscles were higher than the mean carnosine levels in soleus muscles. Carnosine concentrations in soleus muscles and gastrocnemius muscles were significantly higher in the HC group compared to all other groups (p<0.05). Serum triglyceride and LDL-cholesterol concentrations in all of the carnosine-supplemented groups were significantly lower than those of the control group (p<0.05), while HDL-cholesterol levels were significantly higher than those of the control group (p<0.05). Aspartate aminotransferase levels in rats supplemented with carnosine were significantly higher than those of the control group. In conclusion, diets supplemented with high levels of carnosine can increase carnosine concentrations in skeletal muscles, which might contribute to increased exercise capacity. Furthermore, these findings suggest that high levels of dietary carnosine improve the lipid profile of rats by lowering blood LDL-cholesterol and increasing HDL-cholesterol levels.