Abstract: Sugarcane is the main sugar crop in China as well as the world. Sugarcane smut caused by Sporisorium scitamineum is the most important disease in sugarcane production in China and causes great losses every year. Secreted proteins play an important role in establishment of infection and acquisition of nutrients for the pathogen. In the previous study of the genome and transcriptome of S. scitamineum, we identified a β-1,6 glucanase precursor（encoded by Ssa4 p） associated with fungal cell wall degradation. In order to reveal the effects of Ssa4 p on the pathogenicity and secretory proteome of Sporisorium scitamineum, in this study, a gene knockout system was constructed by using CRISPR/Cas9 and T-DNA binary vector. The inactivated mutant of Ssa4 p gene was successfully obtained. The Ssa4 p mutant（Δ35-Ssa4 p） showed reduced coordination ability and pathogenicity with the wild-type strain JG36. By comparison of secretomes of Δ35-Ssa4 p and its parental wide-type strain JG35 by iTRAQ, 1 430 differentially expressed proteins（685 up-regulated and 745 down-regulated） were identified. GO and KEGG analysis revealed that the differentially expressed proteins were mainly located in ribosomes and non-membrane-bounded organelles, which were mainly involved in pathways such as ribosome metabolism, amino sugar and nucleotide sugar metabolism. This study found a β-1,6 glucan precursor-encoding gene was found to impact a large proportion of secretory proteins in a phytopathogenic fungus, broadening our understanding of pathogenicity regulation in pathogenic fungi.