[Objective] This study aimed to clone and characterize a CYP51 homologous gene in Penicillium italicum. [Methods] PCR and Genome walking strategies were used to obtain the gene sequence and its flanking region. Gene structure was analyzed by bioinformatic strategy: Gene transcriptional start site was predicted by NNPP software and TFSEARCH1.3 software was used to predict transcriptional factor binding sites. Homology modeling was performed using human CYP51 protein as the template through the online software SWISS-MODEL. [Results] A CYP51 homologous gene was successfully cloned from Penicillium italicum and named as PiCYP51B. A 3 496 bp sequence including 910 bp 5′ flanking sequence and 834 bp 3′ flanking sequence was obtained. The ORF of PiCYP51B is predicted to encode a protein of 525 amino acids. PiCYP51B contains three introns length 74, 51 and 52 bp, located between 247 bp and 320 bp, 519 bp and 569 bp, 1 635 bp and 1 686 bp respectively. The transcriptional start site is located 458 bp upstream of the initiation codon; the upstream regulatory region not only contain the core structure TATA box (located at 25 bp and 105 bp upstream of the initiation codon), but also contain several transcriptional factor binding sites such as Abd-B, ADR1, AP-4, GATA-1, CdxA, Clox and Oct-1. The proportion of purine is relatively high in the upstream regulatory region. From 387 bp upstream, there are four consecutive heat shot protein transcriptional factor binding sites (HSF); there are three consecutive CdxA transcriptional factor binding sites from 106 bp upstream. To further analysis the structure of PiCYP51B, homology modeling was performed using the online software SWISS-MODEL. [Conclusion] PiCYP51B, being the homologous gene of CYP51 may connect with the resistance to pesticides of P. italicum.