<p>Pancreatic ductal adenocarcinoma (PDAC) is usually treated with gemcitabine (GEM)-based chemotherapy. However, resistance to GEM develops frequently. Aberrant fibroblast growth factor receptors (FGFR) signaling in PDAC is linked to advanced tumor stages and poor prognosis, making it a potential therapeutic target. This study aimed to explore whether inhibition of FGFR by derazantinib could reduce the resistance to GEM in PDAC. Human AsPC-1 and BxPC-3 PDAC cell lines were screened against a panel of FDA-approved compounds to identify the potential drug. Gem-resistant cell lines were subsequently utilized to validate the efficacy of derazantinib. The synergistic interaction between derazantinib and GEM was confirmed through combination-index analysis, clonogenic assays, and apoptosis assays. With RNA-seq, immunohistochemistry, Western blotting, and animal experiments, the effects of derazantinib on the malignant behaviors, signaling pathways in GEM-resistant PDAC cells and tumors were examined. Treatment with derazantinib and GEM synergistically inhibited the malignant behaviors of GEM-resistant PDAC cells and tumor growth by downregulating FGFR2 and FGFR3 expression. RNA-seq revealed upregulated MUC5AC expression in GEM-resistant PDAC, which was attenuated by derazantinib through inhibiting the MAPK and NF-κB signaling. Furthermore, higher levels of FGFR2 and FGFR3 expression were associated with worse survival of PDAC patients and negatively correlated with tumor differentiation. Moreover, the combination of derazantinib and GEM significantly inhibited the growth of GEM-resistant PDAC tumors in vivo. The data highlighted that higher levels of FGFR2, FGFR3, and MUC5AC expression promoted the progression and resistance to GEM in PDAC. Derazantinib treatment enhanced the sensitivity to GEM by attenuating the NF-κB and MAPK pathways to inhibit MUC5AC expression. Therefore, derazantinib may be a promising chemotherapeutic adjuvant for treating PDAC, particularly for patients with GEM resistance.</p>