Understanding the defense mechanisms used by anthracnose-resistant strawberries against Colletotrichum infection is important for breeding purposes. To characterize cell responses to Colletotrichum infection, proteomes from strawberry seedling leaves that had or had not been infected with Colletotrichum fragariae were characterized at different time points post infection by 2-DE and by MALDI-TOF/TOF MS/MS and database-searching protein identification. Mass spectrometry identified 49 differentially expressed proteins with significant intensity differences (>1.5-fold, p<0.05) in mock- and C. fragariae-infected leaves at least at one time point. Notably, 2-DE analysis revealed that C. fragariae infection increased the expression of well-known and novel pathogen-responsive proteins whose expression patterns tended to correlate with physiological changes in the leaves. Quantitative real-time PCR was used to examine the transcriptional profiles of infected and uninfected strawberry leaves, and western blotting confirmed the induction of β-1,3-glucanase and a low-molecular-weight heat shock protein in response to C. fragariae infection. During the late phase of infection, proteins involved in the Calvin cycle and glycolysis pathway had suppressed expression. The abundance changes, putative functions, and participation in physiological reactions for the identified proteins produce a pathogen-responsive protein network in C. fragariae-infected strawberry leaves. Together, these findings increase our knowledge of pathogen resistance mechanisms, especially those found in non-model plant species.