H5N8 subtype highly pathogenic avian influenza viruses (HPAIVs) pose a huge threat to poultry industry and general public health. Our previous study demonstrated that synergistic effect of 283M and 526R in PB2 gene was a critical factor for viral high pathogenicity in mammals. However, the potential pathogenic mechanism of the mutant virus is still unclear. Here, RNA-seq method was used to analyze the global host response of murine lungs after infecting with parental r-JY virus and JY-PB2-I283M-K526R mutant virus. We found that both amounts and the expression levels of host differentially expressed genes (DEGs) were higher in mutant virus-infected mice compared with the group of parental virus. Furthermore, the DEGs mainly related with innate immune response by GO and KEGG analysis. Especially, PB2-I283M-K526R mutation strongly induced a sharp expression of cytokine storm-related genes, including MX1, CXCL10, and IFN-γ, performed by qRT-PCR. We also found that PB2-I283M-K526R mutation accelerated the level of cell apoptosis by heat map analysis of apoptosis-related DEGs in lungs and apoptosis assay in vitro. Taken together, our data demonstrated that PB2-I283M-K526R of H5N8 subtype HPAIV exacerbated the innate immune response and the level of cell apoptosis, which might be a key pathogenic mechanism for the enhanced pathogenicity of mutants in mammals.