Previous microarray analyses revealed that LMO4 is expressed in 3T3‐L1 preadipocytes, however, its roles in adipogenesis are unknown. In the present study, using RT‐PCR sequencing and quantitative real‐time RT‐PCR, we confirmed that LMO4 gene is expressed in 3T3‐L1 preadipocytes and its expression peaks at the early stage of 3T3‐L1 preadipocyte differentiation. Further analyses showed that LMO4 knockdown decreased the proliferation of 3T3‐L1 preadipocytes, and attenuated the differentiation of 3T3‐L1 preadipocytes, as evidenced by reduced lipid accumulation and down‐regulation of PPARγ gene expression. Collectively, our findings indicate that LMO4 is a novel modulator of adipogenesis.