Pancreatic cancer (PC) is one of the most lethal malignant tumors that lacks effective treatment, and gemcitabine-based chemoresistance occurs frequently. Therefore, new therapeutic strategies for PC are urgently needed. Tripartite motif containing 59 (TRIM59) plays an important role in breast and lung cancer chemoresistance. However, the association between TRIM59 and gemcitabine resistance in PC remains unclear. We identified TRIM59 as an innovative E3 ubiquitin ligase that activated Notch signaling in PC. TRIM59 levels were increased in PC and positively correlated with poor prognosis and gemcitabine resistance in PC patients. TRIM59 facilitated gemcitabine resistance in PC cells in vitro and in vivo. TRIM59 interacted with recombination signal binding protein for immunoglobulin kappa J region (RBPJ) and stabilized it by promoting K63-linked ubiquitination. RBPJ transcriptionally upregulated TRIM59 expression, forming a positive feedback loop with TRIM59. We identified a novel TRIM59 inhibitor, catechin, and confirmed that it sensitized PC cells to gemcitabine. TRIM59 conferred gemcitabine resistance in PC by promoting RBPJ K63-linked ubiquitination, followed by activating Notch signaling. Therefore, our study provides a promising target for gemcitabine sensitization in PC treatment.