The objective of the work was to study the optimum conditions of low-fat Cheddar cheese using Lactobacillus plantarum and effect on serum cholesterol in mice. The process parameters were optimized using response surface methodology， and the total cholesterol （TC）， total triglyceride （TG）， high density lipoprotein cholesterol （HDL-C）， low density lipoprotein cholesterol （LDL-C） and atherogenic index （AI） were determined to study the cholesterol lowering effect in vivo. The results showed that the optimum process conditions was 0.02% inoculation amount of starter， 2.00% inoculum amount of probiotic and 2.00% inulin. Compared with hyperlipidemia model， serum TC， TG ， LDL-C and AI levels decreased 21.23%， 25.57%， 39.11% and 35.05% respectively（P<0.05）in group probiotic cheese after 35 days of oral administration（P<0.05）， and TC and TG in liver decreased 37.32% and 17.33%（P<0.05）， but TC and TG in stool increased 37.2% and 30.66%（P<0.05）. Therefore， the low-fat cheese added with Lactobacillus plantarum has certain cholesterol-lowering activity in vivo and sensory quality was acceptable.