Obesity is characterized by excessive accumulation of white adipose tissue. Bromodomain-containing protein 2 (Brd2), which belongs to the bromodomain and extraterminal domain family of proteins, suppresses adipocyte differentiation. DNA methylation is critical for several differentiation processes and possibly in adipocyte differentiation. However, whether DNA methylation regulates the expression of Brd2 is not clear. In our study, we demonstrated that DNA methylation contributes to the regulation of Brd2 expression during pre-adipocyte differentiation. Brd2 mRNA levels were low in pre-adipocytes, increased in early adipocytes, and declined in mature adipocytes. To test whether and how Brd2 expression is regulated by DNA methylation during the differentiation of 3T3-L1 pre-adipocytes to adipocytes, cells were cultured in the presence of the methylation inhibitor 5-aza-2'-deoxycytidine (5-Aza). Pre-adipocytes and adipocytes exposed to 5-Aza exhibited a dose-dependent increase in Brd2 transcription levels, while only mature adipocytes exhibited increased expression of Brd2 protein. Subsequently, we tested the DNA methylation status of the Brd2 promoter region. Bisulfite-sequencing analysis revealed that six CpG sites in two predicted promoters of Brd2 were demethylated in early adipocytes and highly methylated in mature adipocytes. Digestion of bisulfite-converted PCR products of the Brd2 promoter region from 3T3-L1 cells with BstU1 (CGGC) revealed that the demethylation rate of the Brd2 promoter was consistent with Brd2 mRNA expression in differentiating 3T3-L1 cells. In conclusion, DNA demethylation of the Brd2 promoter region induced Brd2 expression during differentiation of 3T3-L1 cells into adipocytes.