ABSTRACTProtein phosphatase complex Nem1/Spo7 plays crucial roles in the regulation of various biological processes in eukaryotes. However, its biological functions in phytopathogenic fungi are not well understood. In this study, genome-wide transcriptional profiling analysis revealed that Nem1 was significantly upregulated during the infection process ofBotryosphaeria dothidea, and we identified and characterized the phosphatase complex Nem1/Spo7 and its substrate Pah1 (a phosphatidic acid phosphatase) inB. dothidea. Nem1/Spo7 physically interacted with and dephosphorylated Pah1 to promote triacylglycerol (TAG) and subsequent lipid droplet (LD) synthesis. Moreover, the Nem1/Spo7-dependently dephosphorylated Pah1 functioned as a transcriptional repressor of the key nuclear membrane biosynthesis genes to regulate nuclear membrane morphology. In addition, phenotypic analyses showed that the phosphatase cascade Nem1/Spo7-Pah1 was involved in regulating mycelial growth, asexual development, stress responses, and virulence ofB. dothidea.IMPORTANCEBotryosphaeria canker and fruit rot caused by the fungusBotryosphaeria dothideais one of the most destructive diseases of apple worldwide. Our data indicated that the phosphatase cascade Nem1/Spo7-Pah1 plays important roles in the regulation of fungal growth, development, lipid homeostasis, environmental stress responses, and virulence inB. dothidea. The findings will contribute to the in-depth and comprehensive understanding of Nem1/Spo7-Pah1 in fungi and the development of target-based fungicides for disease management.