Muscle-invasive bladder cancer (MIBC) remains lethal despite promising oncolytic virotherapy, hindered by tumor-intrinsic resistance. This study aimed to elucidate the molecular basis underlying differential sensitivity to the oncolytic M1 virus in bladder cancer. Bladder cancer cell lines with varying sensitivity to M1 were analyzed for endoplasmic reticulum (ER) stress responses and unfolded protein response (UPR) pathway activation. IRE1α expression was modulated using small interfering RNA and a selective inhibitor. Viral cytotoxicity, replication, and apoptosis were assessed using viability assays, immunofluorescence, electron microscopy, and immunoblotting. antitumor efficacy was assessed using xenografted mice. Clinical relevance was examined using patient-derived cells and survival data from The Cancer Genome Atlas. M1 virus induced ER stress and apoptosis in sensitive cells (e.g., T24, UM-UC-3) supporting viral protein expression, whereas low-sensitivity cells like EJ showed minimal response due to limited viral replication. In moderately sensitive cells, M1 replication led to viral protein accumulation, triggering IRE1α upregulation, which in turn limited further protein buildup and apoptosis. IRE1α inhibition enhanced M1-induced ER stress, apoptotic signaling, and oncolysis without affecting viral replication capacity. , M1 plus STF083010 achieved greater tumor suppression than monotherapy without added toxicity. Analysis of patient-derived cells and TCGA data further revealed downregulation of IRE1α in primary tumors and its potential association with worse prognosis. IRE1α modulates M1-induced viral protein accumulation and cell death. Inhibiting IRE1α enhances ER stress and potentiates the oncolytic effect of M1 virus. Targeting IRE1α may improve M1-based virotherapy outcomes in accessible tumors.