Microsporum canis (M. canis) is a common pathogen that causes tinea capitis and is present worldwide. The incidence of M. canis infection, particularly tinea capitis, has been increasing in china. In our previous studies, family of serine hydrolases 1 (FSH1) was identified as a potential virulence factor in tinea capitis infection caused by M. canis. To determine the function of this gene in M. canis, FSH1 was knocked down using double-stranded RNA interference mediated by Agrobacterium tumefaciens. Reverse transcription-quantitative PCR analysis was used to confirm gene knockdown. Loss of FSH1 expression by RNAi resulted in a minor phenotype alteration, but M. canis pathogenicity in guinea pig cutaneous infection was decreased compared with the wild-type strain. To the best of our knowledge, the present study is the first to demonstrate that FSH1 is associated with macroconidia septa formation and is an important contributor to M. canis virulence. These findings may advance the understanding of the function of the FSH1 gene and provide a foundation for future studies on macroconidia septa formation and pathogenicity of M. canis.