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To investigate a comprehensive transcriptome information of adipogenesis, we assessed global changes in the transcriptional events during 3T3-L1 adipogenesis by RNA-Seq. Compared to the preadipocyte stage (day 0), gene expression profiling demonstrated that 2013 genes were up-regulated, and 2430 genes were down-regulated at the differentiated adipocyte stage (day 13). Among these differentially expressed genes, we found the expression of MSMO1 was down-regulated at day 13, but whether it impacts adipogenesis has not been characterized. Thus, we investigated its role in adipogenesis. Results showed that overexpression of MSMO1 inhibited the differentiation of 3T3-L1, and led to the down-regulated expression of adipogenic marker genes, while knockdown of MSMO1 had totally opposite effects. Furthermore, interaction network model allowed us to validate an unexpected role between MSMO1 and its partner, NSDHL, in regulating adipogenesis, which plays a synergized expression pattern with MSMO1. Our findings indicate that MSMO1 and NSDHL are novel modulators of adipogenesis.