<p>Oncolytic vaccinia virus JX-594 (pexastimogene devacirepvec) selectively replicates in tumour cells and stimulates antitumour immunity; however, its therapeutic potential in gastric cancer (GC) has not been systematically evaluated. In this study, we investigated the antitumour activity of JX-594 across a panel of 49 molecularly characterized GC cell lines and a xenograft mouse model and explored molecular features associated with viral sensitivity. JX-594 induced cytopathic effects (median effective dose, ED₅₀, 0.37 MOI (multiplicity of infection)) in a subset of gastric cancer cell lines and significantly suppressed tumour growth in vivo. Sensitivity to JX-594 varied across molecular subtypes, with microsatellite instability-high (MSI-H) cell lines showing greater susceptibility, whereas Epstein–Barr virus (EBV)–associated cell lines were relatively resistant. Transcriptomic analysis identified candidate biomarkers associated with viral sensitivity, including upregulated genes involved in G-protein signalling (<i>GPR87, GRPR, PRDX5, MX1</i>), interleukin production (<i>IL1A, IL4I1</i>), and vascular activation/dysfunction (<i>OLR1, MMRN2</i>), and downregulation of metastasis-related <i>GAL3ST2</i>. Notably, JX-594 activity was not correlated with TK1 or EGFR expression, suggesting additional determinants of viral susceptibility in GC. These findings provide preclinical evidence that molecular heterogeneity influences the response of GC to oncolytic virotherapy and support further investigation of JX-594 as a potential therapeutic strategy for gastric cancer.</p>