CYP51 of Fungi, known as sterol 14-α-demethylase (P450 14DM) is a key enzyme in the biosynthesis of membrane ergosterol. It will lead to the destruction and loss of function of membrane structure and ultimately to fungal death without CYP51. Based on the 5'-end nucleotide sequence of cyp51, the upstream region of cyp51 was obtained by chromosome walking technique to investigate the regulatory mechanism of cyp51 gene expression. The upstream fragment of 490bp was successfully cloned and sequenced. Analyzed by bioinformatics software, the transcription initiation site of cyp51 was predicted by the NNPP software is located at 134bp in front of ATG, while several cis-acting elements are also analyzed by TFSEARCH 1.3 software. The current study was benefit to further investigating the regulation of cyp51 over-expression and molecular mechanism involved in fungicides resistance of Ustilago maydis in future.