Obesity results in part from altered adipocyte metabolism and enhanced adipogenesis. However, the factors that influence insulin-independent differentiation of preadipocytes in response to excess intake of dietary energy remain poorly understood. Based on our recent finding that LOC66273 isoform 2 (LI2), a gene that encodes a novel Mth938 domain-containing protein, is highly expressed in white adipose tissues, we hypothesized that LI2 plays an important role in adipogenesis. Plasmid pcDNA3.1-LI2 was electroporated into 3T3-L1 preadipocytes to overexpress the LI2 protein. Synthetic siRNA was transfected into 3T3-L1 cells to knockdown endogenous LI2. Using constitutively active and potent siRNA against LI2, we determined cell morphology, cell viability, and adipocytic factors in 3T3-L1 preadipocytes. Our results indicated that LI2 was sufficient to drive preadipocyte differentiation via modulating the phosphorylation level and transcriptional activity of CREB, coincident with expression of several adipogenic regulators and mature adipocyte markers, without insulin treatment. In addition, overexpression of the LI2 protein inhibited preadipocyte growth, whereas knockdown of the LI2 protein resulted in preadipocyte apoptosis via caspase-3 activation during adipogenesis. These results indicated that LI2 might function to switch preadipocytes from proliferation to differentiation and to maintain the viability of preadipocytes during adipogenesis by regulating the caspase-3 pathway. Our findings highlight the importance of LI2 in the formation of new adipocytes, thus helping understand the mechanisms responsible for insulin-independent adipogenesis in mammals.