Mitotic clonal expansion (MCE) is one of the important events taking place at the early stage during 3T3-L1 adipocyte differentiation. To investigate the mechanism underlying this process, we carried out a temporal proteomic analysis to profile the dynamic changes in MCE. Using 8-plex-iTRAQ-2DLC-MS/MS analysis, 3152 proteins were quantified during the initial 28 h of 3T3-L1 adipogenesis. Functional analysis was performed on 595 proteins with maximum or minimum quantities at 20 h of adipogenic induction that were potentially involved in MCE, which identified PI3K/AKT/mTOR as the most relevant pathway. Among the 595 proteins, PKM2 (Pyruvate kinase M2), a patterned protein identified as a potential target gene of C/EBPβ in our previous work, was selected for further investigation. Network analysis suggested positive correlations among C/EBPβ, PIN1, and PKM2, which may be related with the PI3K-AKT pathway. Knockdown of PKM2 with siRNA inhibited both MCE and adipocyte differentiation of 3T3-L1 cells. Moreover, PKM2 was down-regulated at both the mRNA level and the protein level upon the knockdown of C/EBPβ. And overexpressed PKM2 can partially restore MCE, although it did not restore terminal adipocyte differentiation, which was inhibited by siC/EBPβ. Thus, PKM2, potentially regulated by C/EBPβ, is involved in MCE during adipocyte differentiation. The dynamic proteome changes quantified here provide a promising basis for revealing molecular mechanism regulating adipogenesis.