In order to investigate differences in fat metabolism during embryonic development, a comparative proteomics strategy was employed using Arbor Acres (AA) and San Huang (SH) broiler chickens with different growth and fat deposition characteristics. These birds were floor-reared and fed identical diets, and embryonic livers were collected from AA and SH chicken embryos on days 9, 14 and 19 of incubation and hatching. Proteins were extracted and fractionated by two-dimensional electrophoresis (2-DE), Neuhoff's colloidal Coomassie Blue G-250 staining was carried out, and stained gels were scanned and analyzed using PDQuest7.3 software (Bio-Rad). In-gel trypsin digestion of the differential protein spots and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) were subsequently assessed. Peptide mass fingerprinting of the differentially expressed proteins was performed using the server from MASCOT or either Prospector or ProFound, and 37 proteins were successfully identified. In the present study, embryo and liver weights showed a trend toward enhanced growth during embryonic development. Of the 37 identified differential proteins, phosphoenolpyruvate carboxykinase (PEPCK), apolipoprotein A-I (Apo A-I), fatty acid-binding protein (L-FABP) and 3-hydroxy-3-methylglutaryl-Coenzyme A synthase (HMG-CoA synthase) were up-regulated in SH chickens to a greater extent than they were in AA chickens. These observations suggest that the lipid metabolic proteins and enzymes are inherent characteristics that contribute to the apparent differences in fat deposition between the two strains.