Cholangiocarcinoma (CCA) is an aggressive cancer originating from bile duct epithelium. Surgical resection remains the primary curative treatment for CCA. However, most CCA patients are diagnosed at an advanced stage, which limits the applicability of surgical resection. Gemcitabine is widely used as a first-line chemotherapeutic agent for unresectable CCA. Its efficacy is often compromised by the development of drug resistance, which leads to poor clinical outcomes and low survival rates of CCA patients. At present, the mechanisms underlying gemcitabine resistance in CCA remain unclear. This review aimed to comprehensively summarize the current knowledge on the molecular mechanisms underlying gemcitabine resistance in CCA and highlight emerging therapeutic strategies that may overcome this resistance. Gemcitabine resistance arises through multiple mechanisms, including reduced drug uptake and increased efflux, impaired drug activation, enhanced DNA repair, apoptosis evasion, aberrations in cell-cycle progression, induction of epithelial-mesenchymal transition, metabolic reprogramming, alteration of tumor, and activation of oncogenic pathways contributes to gemcitabine resistance. A deeper understanding of gemcitabine resistance mechanisms highlights the need for combining gemcitabine with pathway-specific inhibitors, which hold promise for overcoming resistance and improving patient outcomes.