Pear ring rot disease, the pathogen of Botryosphaeria dothidea causes significant threat to the healthy development of the pear industry, therefore the exploration of disease-resistant gene resources is crucial for disease prevention and control. Members of the R2R3-MYB subfamily play important roles in regulating pathogen resistance in plants, however the gene function in regulating host resistance in pear remains unclear. In this study, the role of PcMYB44 were investigated in regulating host resistance disease in pear calli using both forward and reverse genetic approaches. Overexpression of PcMYB44 positively regulates the disease resistance, whereas knockout of PcMYB44 results in a phenotype with decreased resistance. Our results further demonstrated that PcMYB44 could directly affect lignin content and resistance to fungal diseases by regulating the PcmiR397-PcLACs module and lignin biosynthesis gene expression levels. Additionally, overexpressing PcMYB44 also elevated expression levels of key genes of JA/SA/ET pathway. The obtained results revealed that PcMYB44 regulated host resistance to ring rot disease through synergistic regulation the lignification and activating disease-resistance gene expression of JA/SA/ET defense pathways as a underlying secondary mechanism, which provide valuable genetic resources for molecular breeding for disease resistance.