The pathogenic fungus Neoscytalidium dimidiatum (Nd) is the causal agent of pitaya canker and causes significant yield losses. The mechanism by which Nd invades pitaya stems remains largely unknown. Here, quantitative proteomic analysis was employed to investigate pitaya immune responses against Nd infection. A total of 2766 proteins including 244 differentially expressed proteins (DEPs) were identified during the infection cycle. Bioinformatics analysis indicated that the DEPs were associated with photosynthesis, phytohormone activity, reactive oxygen species (ROS) homeostasis and pathogenic defense responses. This study demonstrates that comparative proteomics is an effective strategy for providing new perspective on biotic stress from fungal pathogens in pitaya.