OBJECTIVE The aim of this study was to gain insight into those proteins that might be involved in the early stages of liver fat accumulation as a consequence of a different fat versus simple sugar dietary intake. METHODS Forty-five male Wistar rats were randomly distributed into four dietary groups: a starch-rich control diet (CD; n = 10), a high-fat diet (n = 12), a high-sucrose diet (n = 11), and a high-fat sucrose diet (HFSD; n = 12) for 5 weeks. A comparative analysis by 2D-DIGE and LC-ESI-MS/MS was performed to characterize the liver protein expression profiles due to the three obesogenic diets. RESULTS Ten out of 17 proteins whose expression levels were altered by >1.25-fold were identified. Four proteins (Hspa8, Hspa9, Ca3, and Cat) were differentially expressed after the HFSD period compared to CD. The heat shock proteins (Hspa8 and Hspa9) resulted significantly downregulated in liver from rats fed HFSD versus CD (p < 0.05). The results were confirmed by Western blot. CONCLUSIONS This descriptive study might be useful for further studies aiming at understanding the mechanisms by which diets rich in both fat and sugar affect the initiation of hepatic steatosis.