Background/Objectives: Pancreatic ductal adenocarcinoma is a lethal malignancy, necessitating an understanding of its molecular mechanisms for the development of new therapeutic strategies. The tight junction protein claudin-1, known to influence cellular functions in various cancers and is considered a therapeutic target, remains unclear in pancreatic cancer. Methods: This study assessed claudin-1 expression in resected pancreatic cancer samples, public databases, and pancreatic cancer cell lines. Claudin-1 knockout with CRISPR/Cas9 on poorly differentiated pancreatic cancer cell lines and a proteome analysis were performed to investigate the intracellular mechanisms of claudin-1. Results: Claudin-1 was markedly overexpressed in pancreatic ductal adenocarcinoma and intraepithelial neoplasia compared to normal ducts, and high claudin-1 levels were an independent predictor of poor prognosis. Claudin-1 knockout diminished cell proliferation, migration, invasion, and chemoresistance in pancreatic ductal adenocarcinoma. Proteome analysis revealed the significant downregulation of aldo-keto reductase family proteins (AKR1C2, AKR1C3, and AKR1B1) in claudin-1 knockout cells, which are linked to metabolic pathways. Aldo-keto reductase knockdown reduced chemoresistance, proliferation, and invasion in these cell lines. Conclusions: These findings indicate that the abnormal expression of claudin-1 promotes tumor progression and drug resistance through its interaction with aldo-keto reductase proteins, highlighting claudin-1 and aldo-keto reductase family proteins as potential biomarkers and therapeutic targets for pancreatic cancer.