As a member of the basic helix-loop-helix (bHLH) family of transcription factors, Bhlhe40 plays an important role in the regulation of adipogenesis, myogenesis and the development of the metabolic syndrome. In this study, we investigated the tissue expression patterns and differences of bovine Bhlhe40 gene during three developmental stages. The result showed that Bhlhe40 was ubiquitously expressed in most tissues of cattle. In addition, the expression of bovine Bhlhe40 gene showed a decreasing trend in skeletal muscle and fat during the developmental stages. Then, two novel mutations (EX5 + 114 T>C, L166P and EX5 + 733 A > G, S372S) of bovine Bhlhe40 gene were identified by BamHI and ApaI ACRS PCR-RFLP in 1226 individuals from five indigenous cattle breeds in China. Frequencies of allele C at the BamHI locus varied from 0.210 to 0.340 among breeds, while frequencies of allele C at the ApaI locus varied from 0.030 to 0.114. Association analysis revealed no significant association of the ApaI locus with growth traits in Nanyang breed. However, the BamHI locus was significantly associated with the average daily gain at 18 months in Nanyang cattle (P< 0.05). This study aimed to lay the groundwork to further study of Bhlhe40 function at myogenesis and adipogenesis in bovine and should significantly contribute to cattle breeding and genetics through the MAS program.