TonEBP belongs to the Rel family of transcription factors and plays important roles in inflammation as well as kidney homeostasis. Recent studies suggest that TonEBP expression is also involved in differentiation of several cell types such as myocytes, chondrocytes, and osteocytes. In this study, we investigated the roles of TonEBP during adipocyte differentiation in 3T3-L1 cells. TonEBP mRNA and protein expression was dramatically reduced during adipocyte differentiation. Sustained expression of TonEBP using an adenovirus suppressed the formation of lipid droplets as well as the expression of FABP4, a marker of differentiated adipocytes. TonEBP also inhibited the expression of PPARγ, a known master regulator of adipocytes. RNAi-mediated knock down of TonEBP promoted adipocyte differentiation. However, overexpression of TonEBP did not affect adipogenesis after the initiation of differentiation. Furthermore, TonEBP expression suppressed mitotic clonal expansion and insulin signaling, which are required early for adipocyte differentiation of 3T3-L1 cells. These results suggest that TonEBP may be an important regulatory factor in the early phase of adipocyte differentiation.