Aim To compare the liver protein profiles of hypercholesterolemic and normal mice by using the technology of proteomics. Methods The hypercholesterolemic mice were obtained after feeding atherogenic diet for 14 weeks. Protein extractions from mice livers were separated by two-dimensional electrophoresis and the gels were analysed with image analysis software. The differentially expressed proteins were identified primarily by mass spectrometry and then confirmed by comparing with the nice gel image in protein database Swiss Prot. Results The hypercholesterolemic mouse model has been successfully prepared. The total cholesterol levels in plasma and liver of hypercholesterolemic mice increased significantly. The protein extractions separated by two-dimensional electrophoresis have got high resolution and reproducibility. Sixteen differentially expressed protein spots( > 2 fold) have been found and 8 of which were identified as major urinary proteins (MUPs), carbonic anhydrase Ⅲ and Glutathione S-transferase P2. Conclusion The under-expression of MUPs, carbonic anhydrase Ⅲ and Glutathione S-trans ferase P2, which regulated by androgens, may be related to diet-induced hypercholesterolemia.