Malignant tumor cells undergo profound metabolic reprogramming, manifested by a markedly enhanced capacity for the uptake of specific metabolic substrates, most notably glutamine (Gln). This metabolic adaptation not only provides essential precursors to support aberrantly active glycolysis and mitochondrial oxidative phosphorylation but also disrupts the homeostatic balance of carbohydrate, lipid, and amino acid metabolism, thereby directly contributing to the adaptive resistance of tumor tissues to chemotherapeutic agents. Despite its significance, a systematic understanding of how tumor cells modulate glutamine metabolic plasticity to evade drug-induced cytotoxicity remains elusive. Furthermore, there is a notable lack of in-depth analyses addressing the context-dependent variability of glutamine metabolism across different cancer types and microenvironments, as well as the underlying causes of recurrent failures in clinical translation. This review systematically delineates the key regulatory roles and mechanistic pathways through which glutamine metabolism drives the acquisition of drug resistance in malignancies. For the first time, we propose a three-dimensional resistance framework—termed the "glutamine metabolism–microenvironment–immune evasion" model—and identify three major bottlenecks inherent in current targeted therapeutic strategies. Collectively, this review aims to establish a theoretical foundation and provide methodological insights to inform and improve clinical treatment strategies for drug-resistant malignant tumors.