<p>Cancer stem cells (CSCs) are key drivers of cancer progression and therapeutic resistance. Long non-coding RNAs (lncRNAs) have emerged as critical regulators of CSC properties. The aim of this study was to investigate the role of MIR4435-2HG in regulating CSC characteristics, tumorigenesis, and chemoresistance in pancreatic cancer. Functional assays were conducted to evaluate CSC self-renewal, tumorigenic potential, and chemoresistance in pancreatic cancer cells with altered expression of MIR4435-2HG. RNA interference (RNAi) was employed to knock down MIR4435-2HG, and a STAT1 reintroduction model was established to examine downstream signaling pathways. The role of miR-1252-5p as a competing endogenous RNA was also explored. Overexpression of MIR4435-2HG significantly enhanced CSC self-renewal and tumorigenic potential, whereas silencing MIR4435-2HG notably diminished these properties. Mechanistically, MIR4435-2HG promoted STAT1 expression by sponging miR-1252-5p, thereby enhancing CSC stemness and tumorigenesis. Moreover, depletion of MIR4435-2HG sensitized pancreatic cancer cells to gemcitabine-induced growth inhibition and ferroptosis. Reintroduction of STAT1 restored gemcitabine resistance in MIR4435-2HG-deficient cells. Our findings demonstrate that MIR4435-2HG plays a critical role in pancreatic cancer progression by modulating CSC properties and chemoresistance through the MIR4435-2HG/miR-1252-5p/STAT1 axis. Targeting MIR4435-2HG presents a promising therapeutic approach to regulate CSCs and improve the efficacy of chemotherapy in pancreatic cancer.</p>