Oncolytic virotherapy is an emerging treatment modality that uses replication-competent viruses to destroy cancer cells. M1 is a naturally occurring alphavirus ( Togaviridae ) which shows potent oncolytic activities against many cancers. Accumulation of unfolded proteins during virus replication leads to a transcriptional/translational response known as the unfolded protein response (UPR), which might counteract the antitumor effect of the oncolytic virus. In this report, we show that either pharmacological or biological inhibition of IRE1α or PERK, but not ATF6, substantially increases the oncolytic effects of the M1 virus. Moreover, inhibition of IRE1α blocks M1 virus-induced autophagy, which restricts the antitumor effects of the M1 virus through degradation of viral protein, in glioma cells. In addition, IRE1α suppression significantly increases the oncolytic effect of M1 virus in an orthotopic glioma model. From a molecular pathology study, we found that IRE1α is expressed at lower levels in higher-grade gliomas, suggesting greater antitumor efficacy of the oncolytic virus M1. Taken together, these findings illustrate a defensive mechanism of glioma cells against the oncolytic virus M1 and identify possible approaches to enhance the oncolytic viral protein accumulation and the subsequent lysis of tumor cells. IMPORTANCE Although oncolytic virotherapy is showing great promise in clinical applications, not all patients are benefiting. Identifying inhibitory signals in refractory cancer cells for each oncolytic virus would provide a good chance to increase the therapeutic effect. Here we describe that infection with the oncolytic virus M1 triggers the unfolded protein response (UPR) and subsequent autophagy, while blocking the UPR-autophagy axis significantly potentiates the antitumor efficacy of M1 in vitro and in vivo . A survey of cancer tissue banks revealed that IRE1α, a key element in the UPR pathway, is commonly downregulated in higher-grade human gliomas, suggesting favorable prospects for the application of M1. Our work provides a potential predictor and target for enhancement of the therapeutic effectiveness of the M1 virus. We predict that the mechanism-based combination therapy will promote cancer virotherapy in the future.