Background <p>Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are widely used in the treatment of estrogen receptor–positive (ER⁺) breast cancer; however, the metabolic adaptations induced by CDK4/6 inhibition remain incompletely defined. In ER⁺ breast cancer, estrogen signaling plays a central role in coordinating cell cycle progression and metabolic programs that support tumor growth. Glycolytic flux is regulated at the level of phosphofructokinase-1 (PFK1) through the inducible enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), which is transcriptionally regulated by estrogen receptor signaling and has been shown to promote glycolysis and proliferation in ER⁺ breast cancer cells. Yet, how CDK4/6 inhibition intersects with estrogen-regulated glycolytic control to rewire glucose utilization in ER⁺ breast cancer has not been explored.</p> Methods <p>Glucose metabolism was assessed using extracellular flux analysis, untargeted metabolomics, and stable isotope tracing with uniformly labeled ¹³C-glucose in ER+ breast cancer cell lines. In vivo metabolic tracing was performed following bolus administration of [U-¹³C]-glucose. The effects of pharmacologic PFKFB3 inhibition, alone and in combination with CDK4/6 inhibitors, were evaluated in vitro and in patient-derived xenograft (PDX) models. Statistical analyses were performed using appropriate tests with correction for multiple comparisons where applicable.</p> Results <p>CDK4/6 inhibition increased glycolytic flux, as evidenced by elevated basal and compensatory glycolysis, accumulation of early glycolytic intermediates, and increased ¹³C labeling of fructose 1,6-bisphosphate. PFKFB3 deficiency significantly attenuated the CDK4/6 inhibitor-induced increase in glycolytic flux. Despite increased glycolysis, stable isotope tracing revealed markedly reduced incorporation of glucose-derived carbon into nucleotide biosynthesis and lipid-associated metabolites, consistent with reduced anabolic demand during G1 cell cycle arrest. In vivo glucose tracing demonstrated a dissociation between increased glycolytic flux and downstream biosynthetic utilization. Pharmacologic inhibition of PFKFB3 imposed additional constraints on glucose utilization and significantly enhanced the antitumor efficacy of CDK4/6 inhibition in PDX models.</p> Conclusions <p>CDK4/6 inhibition rewires glucose metabolism in ER+ breast cancer by increasing glycolytic flux while limiting downstream glucose utilization, resulting in heightened reliance on regulated glycolytic control to maintain metabolic homeostasis during cell cycle arrest. Disruption of this adaptive metabolic state through PFKFB3 inhibition enhances the antitumor effects of CDK4/6 inhibition and supports the therapeutic potential of targeting glycolytic regulation in combination with CDK4/6 inhibitor-directed therapies.</p>