In order to explore the mechanism of DNA methylation on gene expression regulation of loach Misgurnus anguillicaudatus, as well as other teleosts, the 5"-flanking sequence of DNA methyltransferase gene family dnmts in M. anguillicaudatus were cloned, and spatiotemporal expression of dnmts were analyzed. Obvious differences were observed in the subcellular localization and N-terminal regulatory domain, the regulatory domain of dnmt1 includes DMAP, PBD, CXXC and BAH conserved domains, while dnmt3s contain PWWP, ADD or CH domains, indicating that there is a large functional differentiation between dnmt1 and dnmt3s. And the aforementioned inference was further confirmed by the phylogenetic tree constructed based on amino acid sequence. Using hiTAIL-PCR method, the 5"-flanking sequence of each dnmt was cloned, and the typical function element TATA-box, as well as some binding sites of transcription factors related to embryonic development, growth and reproduction such as CREB、Egr-1、Pit-1 were predicted on the sequences. Expression levels of the 4 dnmts were dynamically changed and correlated with the developmental stages, for example, the dnmt1 was high expressed in the rapid cleavage stage from cleavage stage to blastocyst stage, while dnmt3aa and dnmt3ab were high expressed in the late embryonic stage of organ differentiation. In adult fish, dnmt1 was highly expressed in the gonads; dnmt3aa and dnmt3ab highly expressed in brain, while dnmt3b highly expressed in gonads and muscles. In addition, the expression of dnmts showed obvious sex dimorphism. For example, the expression levels of dnmt1 and dnmt3b in ovary were significantly higher than those in testis, while dnmt3aa and dnmt3ab were in opposite manner. Furthermore, dnmt3aa was expressed in both germ cells and somatic cells of the ovary, while dnmt3ab was only expressed in somatic cells. The results above all indicate that there is functional differentiation among dnmts members and they play important functions in the corresponding tissues.