Background <p>Gemcitabine is the primary chemotherapeutic agent used as a first-line treatment for pancreatic cancer (PC), underscoring its pivotal role in cancer treatment. This drug induces apoptosis through multiple mechanisms, with the disruption of cellular pyrimidine metabolism being a crucial mode of action. However, gemcitabine resistance significantly limits the long-term efficacy of PC treatments. Our study aimed to identify potential targets for gemcitabine sensitization.</p> Methods <p>We identified a gene set associated with pyrimidine metabolism through cluster analysis of The Cancer Genome Atlas Pancreatic Adenocarcinoma database. Intersecting this set of differentially upregulated genes in PC with normal tissues revealed inhibin βA (<i>INHBA</i>) as a gene related to pyrimidine metabolism. Quantitative reverse transcription-polymerase chain reaction, western blotting, and immunohistochemistry analyses confirmed <i>INHBA</i> overexpression in PC cells. INHBA’s effects of INHBA on cell proliferation and chemoresistance in PC cells were validated in vitro using a colony counting kit-8 assay, drug sensitivity analysis, EdU incorporation, flow cytometry, and colony formation assay. The proliferation and chemoresistance promotion by INHBA in PC cells was validated in vivo using a mouse xenograft model. Downstream targets of INHBA were identified using immunoprecipitation mass spectrometry.</p> Results <p>INHBA, a pyrimidine metabolism-associated gene, was highly expressed in PC, and its expression correlated with gemcitabine resistance. INHBA interacts with cytidine triphosphate synthase 1 (CTPS1) and competitively inhibits SMAD Specific E3 Ubiquitin Protein Ligase 1 mediated ubiquitination, enhancing CTPS1 stability and promoting pyrimidine metabolism.</p> Conclusion <p>INHBA promotes gemcitabine resistance in PC by stabilizing CTPS1 and enhancing pyrimidine metabolism, making it a potential target for chemosensitization in PC.</p> Graphical abstract <p></p>