Oncolytic viruses (OVs), known for their cancer-killing characteristics, overturn tumor-associated defects in antigen presentation through the MHC class I pathway and induce protective neo antitumor CD8 T cell responses. Nonetheless, whether OVs shape the tumor MHC-I ligandome remains unknown. Here, we investigated if an OV induces the presentation of novel MHC I-bound tumor antigens (termed tumor MHC-I ligands). Using comparative mass spectrometry (MS)-based MHC-I ligandomics, we determined differential tumor MHC-I ligand expression following treatment with oncolytic reovirus in a murine ovarian cancer model. In vitro we found that reovirus induces the presentation of tumor MHC-I ligands in cancer cells. Concurrent multiplexed quantitative proteomics revealed that the changes in tumor MHC-I ligand presentation were mostly independent of reovirus-induced alterations of their source proteins. In an in vivo model, tumor MHC-I ligands were induced by reovirus in tumors but also, more importantly, analysis of spleens (a source of antigen-presenting cells) showed exclusive induction of most MHC-I ligands occurred in tumor-bearing mice. Finally, IFNγ assays demonstrated immunogenicity of the reovirus-induced MHC-I ligands. OV-induced MHC-I responses may be exploited in combinatorial approaches to promote the efficacy of cancer immunotherapies.