In this study, the role of WSC1 in the infection of pear fruit by Penicillium expansum was investigated. The WSC1 gene was knocked out and complemented by Agrobacterium-mediated homologous recombination technology. Then, the changes in growth, development, and pathogenic processes of the knockout mutant and the complement mutant were analyzed. The results indicated that deletion of WSC1 slowed the growth rate, reduced the mycelial and spore yield, and reduced the ability to produce toxins and pathogenicity of P. expansum in pear fruits. At the same time, the deletion of WSC1 reduced the tolerance of P. expansum to cell wall stress factors, enhanced antioxidant capacity, decreased hypertonic sensitivity, decreased salt stress resistance, and was more sensitive to most metal ions. Our results confirmed that WSC1 plays an important role in maintaining cell wall integrity and responding to stress, toxin production, and the pathogenicity of P. expansum.