The expression levels of 97 unigenes encoding heat shock proteins of Litopenaeus vannamei was scanned, and ten of them were significantly induced by white spot syndrome virus (WSSV). Among these genes, heat shock 70 kDa protein cognate 5 (LvHSC70-5) was upregulated to the highest extent and subjected to further studies. Subcellular localization assay revealed that LvHSC70-5 was located in the mitochondria. Aside from WSSV infection, unfolded protein response activation and thermal stress could also upregulate LvHSC70-5. Results of reporter gene assay demonstrated that promoter of LvHSC70-5 was activated by L. vannamei heat shock factor protein 1, activating transcription factor 4 and thermal stress. A decrease in the expression of LvHSC70-5 could reduce the aggregation of proteins in hemocytes and the cumulative mortality of WSSV-infected L. vannamei. LvHSC70-5 in L. vannamei hemocytes was upregulated by mild thermal stress. In addition, mild thermal stress, decreased the copy number of WSSV in shrimp muscle and the cumulative mortality of WSSV-infected L. vannamei. Therefore, collecting results suggested that LvHSC70-5 should be involved in WSSV toleration of shrimp L. vannamei.