Background <p>Metabolic reprogramming is a hallmark of rapid tumor cell progression. Cellular cyclin-dependent kinase 6 (CDK6) has been reported to exhibit abnormal expression in oral squamous cell carcinoma (OSCC). However, the detailed functional role of CDK6 in OSCC glycolytic reprogramming is still unknown and requires further investigation. Studies have demonstrated that miR-181a-5p plays a significant role in the malignant progression of OSCC. Therefore, this study aimed to elucidate the molecular mechanisms underlying the role of the miR-181a-5p/CDK6 axis during glycolysis in OSCC.</p> Methods <p>The expression and functional testing of miR-181a-5p and CDK6 were conducted both in vivo and in vitro, encompassing cell proliferation, migration, and glycolysis-related metabolites. The association between miR-181a-5p expression and clinicopathological factors as well as the overall survival was analyzed. A dual-luciferase reporter assay was used to confirm the relationship between miR-181a-5p and CDK6. Quantitative real-time polymerase chain reaction(qRT-PCR) and western blotting were performed to detect gene and protein expression, respectively.</p> Results <p>CDK6 knockdown significantly inhibited OSCC cell proliferation, migration, glucose consumption, lactate, ATP production, and downregulated the expression or catalytic activities of HK2, PFKM, PKM2, LDHA, PHGDH, PSAT1, but not G6PD. Overexpressed miR-181a-5p increased overall survival (OS). CDK6 was a direct target of miR-181a-5p. CDK6 overexpression phenotypically rescued the inhibitory effect of miR-181a-5p on the proliferation, migration, and glycolytic reprogramming of OSCC cells.</p> Conclusions <p>These findings underscore the critical role of CDK6 in the OSCC progression and provide the first evidence that miR-181a-5p regulates CDK6, thereby suppressing glycolysis and the serine-glycine biosynthesis pathway (SSP) in OSCC. However, miR-181a-5p attenuates the pentose phosphate pathway (PPP) via CDK6-independent targets.</p>