Liver hepatocellular carcinoma (LIHC) and head and neck squamous cell carcinoma (HNSC) are aggressive cancers with high morbidity and mortality. Identifying reliable biomarkers for early diagnosis, prognosis, and therapeutic targeting is crucial. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is traditionally known as a glycolytic enzyme but is increasingly recognized for its role in cancer biology. This study investigates the expression and promoter methylation of GAPDH in LIHC and HNSC, utilizing data from the UALCAN and GEPIA2 databases. Our results show that GAPDH expression is significantly upregulated in primary tumor tissues compared to normal tissues in both LIHC and HNSC. This upregulation is associated with reduced promoter methylation, suggesting hypomethylation as a potential mechanism. Furthermore, the validation of these findings across different cancer stages indicates that GAPDH expression increases from stage I to stage III in both cancer types, with a decrease in stage IV for LIHC and high variability in HNSC. Promoter methylation levels are consistently lower in tumor samples compared to normal tissues, with increased gene body methylation. Mutational analysis via the cBioPortal database reveals that GAPDH alterations are more frequent in HNSC than in LIHC, though they do not significantly impact overall survival. Kaplan-Meier survival curves from the KM plotter tool indicate that high GAPDH expression correlates with worse overall survival and relapse-free survival in LIHC, whereas in HNSC, high GAPDH expression affects overall survival but not relapse-free survival. Protein-protein interaction network and gene enrichment analyses highlight GAPDH’s involvement in metabolic processes, protein synthesis, and extracellular interactions. Drug sensitivity analysis shows a positive correlation between GAPDH expression and resistance to multiple therapeutic agents, suggesting GAPDH as a potential biomarker for predicting drug sensitivity. Overall, these findings underscore the potential of GAPDH as a diagnostic, prognostic, and therapeutic target in LIHC and HNSC.