BACKGROUND: Ribosome-inactivating proteins (RIPs) are a group of proteins known to inhibit protein synthesis and contribute to plant defense responses. Although the antiviral properties of various RIPs have been demonstrated, the antiviral potential of tritin, a type 1 RIP from bread wheat (Triticum aestivum L.), remains unexplored. This study aimed to investigate the antiviral activity of recombinant tritin against zucchini yellow mosaic virus (ZYMV) in zucchini (Cucurbita pepo L.) plants. METHODS: The tritin gene was isolated from the wheat cultivar Kutluk-94, cloned into the pETDuet-1 expression vector, and expressed in Escherichia coli BL21 (DE3) cells. Following induction, recombinant tritin was purified using Ni-NTA affinity chromatography. Antiviral activity was assessed by measuring morphological parameters, disease severity, and the expression levels of the ZYMV Coat Protein (CP) gene and the Pathogenesis-Related 1 (PR1) through quantitative real-time PCR. RESULTS: Tritin-treated plants exhibited significantly lower ZYMV-CP gene expression compared to virus-inoculated controls at 3- and 15- days post-inoculation. Furthermore, PR1 gene expression was upregulated in response to tritin application, suggesting the activation of systemic defense pathways. Morphological assessments revealed dose-dependent phytotoxic effects, including reductions in chlorophyll content and plant growth at higher tritin concentrations. CONCLUSION: This study represents the first report in demonstrating that recombinant tritin exhibits antiviral activity against ZYMV, reducing viral replication and enhancing defense gene expression in zucchini plants. However, the phytotoxic effects observed at higher concentrations suggest the need for dose optimization before agricultural application. These findings provide a promising basis for the development of RIP-based antiviral strategies to improve crop tolerance.