BACKGROUND: Oncolytic virus therapy (OVT) represents a promising frontier in cancer treatment. Despite its efficacy in clinical trials, variability in patient response, particularly resistance development, highlights the need for tailored therapeutic strategies. METHODS: The Inositol Hexakisphosphate Kinase 2 (IP6K2) gene knock out was carried by CRISPR/Cas9 system. The evaluation of biomarkers of apoptosis and relevant pathways was conducted to be assessed. Attachment assay was conducted to verify the binding ability of virus to the host cells. Cell proliferation and apoptosis was assessed. Subcutaneous xenograft model was used to evaluate IP6K2 knock out influence in vivo. cBioPortal and TCGA database were applied to analyze genomic alterations in pan-cancer. RESULTS: IP6K2 was essential for effective Herpes Simplex Virus Type1 (HSV-1) replication and subsequent cell apoptosis, acting through the tumor Protein p53 (p53) and Cyclin-Dependent Kinase Inhibitor 1 A (p21) signaling axis. The tumor model demonstrated that tumors lacking IP6K2 exhibited resistance to HSV-1 oncolysis, resulting in diminished therapeutic outcomes. Analysis of cBioPortal and TCGA databases corroborated the potential resistance stemming from IP6K2 mutations across various cancer types, underscoring the necessity for pre-treatment IP6K2 status assessment. CONCLUSIONS: This study underscores the role of IP6K2 as potential markers of resistance, which opens avenues for precision medicine approaches in OVT.