Corynespora leaf spot (CSL), caused by Corynespora cassiicola, has become one of the most important foliar diseases of cultivated cucumber. However, the defense mechanisms of cucumber plants in response to C. cassiicola are still poorly understood. Here, proteins from resistant plants were analyzed using isobaric tags for relative and absolute quantification (iTRAQ). A total of 286 differentially expressed proteins were identified (P<0.05, ratio>1.2 or <0.83) at 6 and 24 h after pathogen inoculation in the resistant cucumber cultivar Jinyou 38. Some of the early responses to C. cassiicola infection were revealed, and four vital clues regarding the resistance of Cucumis sativus to cucumber CLS were discovered. First, the proteomic approach revealed the modulation of signaling pathways in resistant cucumber plants in response to C. cassiicola infection. Second, the plant immune system recognizes the pathogen and initiates the expression of basal immune response proteins, including those related to defense and stress responses, signal transduction, cell metabolism and redox regulation. Third, the common stress pathways were activated by C. cassiicola; in particular, mildew resistance locus O (MLO) proteins played a crucial role in the prevention of CLS. Fourth, the rapid activation of the carbohydrate and secondary metabolic pathways, the modification and reinforcement of cell walls, and the adjustment of the apoplectic environment to high-stress conditions were crucial in cucumber resistance to CLS disease. Overall, our data increase the knowledge of incompatible interactions between plants and pathogens and provide new insight into the contribution of molecular processes in cucumber to disease resistance.