Our previous study found that the expression of HSP17.4 is closely related to the occurrence of strawberry anthracnose. In this study, we used virus-mediated gene silencing technology and found that the HSP17.4 gene-silenced ‘Sweet Charlie’ plants were more susceptible to Colletotrichum gloeosporioides than the wild type ‘Sweet Charlie’, and the level of infection was even higher than that of the susceptible cultivar ‘Benihopp’. The results of differential quantitative proteomics showed that after infection with the pathogen, the expression of the downstream response genes NPR1, TGA and PR-1 of the salicylic acid (SA) signaling pathway was fully upregulated in the wild-type ‘Sweet Charlie’, and the expression of the core transcription factor MYC2 of the jasmonic acid (JA) pathway was significantly down-regulated. The expression of the proteins encoded by these genes did not change significantly in the HSP17.4-silenced ‘Sweet Charlie’, indicating that the expression of HSP17.4 activated the up-regulation of downstream signals of SA and inhibited the JA signal pathway. The experiments that used SA, methyl jasmonate and their inhibitors to treat plants provide additional evidence that the antagonism between SA and JA regulates the resistance of strawberry plants to C. gloeosporioides.