Fungal pathogens cause severe postharvest decay of fruit, while Botrytis cinerea is one of the most notorious among these fungal pathogens. Consequently, a mechanistic dissection of B. cinerea pathogenesis has important significance for scientific research, particularly the key factors regulating B. cinerea virulence at early infection stage. This study performed proteomic profiling for B. cinerea conidial suspension after optimization for the on planta experimental system. The results revealed that a total of 219 proteins were involved in the infection of host plants by B. cinerea. They were typically secretory proteins and mainly categorized as carbohydrate hydrolyzing enzymes, cell wall degrading proteins, redox-related proteins, proteases, and proteins with unknown functions. Further phenotypic analysis on the deletion mutants demonstrated that these proteins differentially modulated the growth, conidiation and virulence of B. cinerea on fruit, respectively. Moreover, the proteins were significantly up-regulated during the infection of fruit tissues, displaying different patterns as compared to those in cultivated mycelia. These results may provide references for dissecting the mechanisms underlying B. cinerea pathogenesis and exploring molecular targets for controlling grey mold.