Adipose tissue is an important endocrine organ involved in the control of whole body energy homeostasis and insulin sensitivity. Considering the increased incidence of obesity and obesity-related disorders, including diabetes, it is important to understand thoroughly the process of adipocyte differentiation and its control. Therefore, we performed a differential proteome mapping strategy using two-dimensional gel electrophoresis combined with peptide mass fingerprinting to identify intracellular proteins that are differentially expressed during adipose conversion of 3T3-L1 pre-adipocytes in response to an adipogenic cocktail. In the current study, we identified 46 differentially expressed proteins, 6 of which have not been addressed previously in 3T3-L1 cell differentiation. Notably, we found that phosphoribosyl pyrophosphate synthetase (PRPS), a regulator of cell proliferation, was preferentially expressed in pre-adipocytes than in fully differentiated adipocytes. In conclusion, our results provide valuable information for further understanding of the adipogenic process.