Background <p>Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer, with high mortality and limited treatment approaches. This paper explores the function of TAL1 in NSCLC progression and glycolysis and its mechanism.</p> Methods <p>Bioinformatics analysis screened out TAL1 and the upstream and downstream molecules. MTT, EdU, wound healing assay, Transwell assay, and TUNEL were utilized to detect the malignant phenotype of A549 and H460 cells. Western blot analysis was conducted to detect the expression of the proliferation-associated protein (Ki67), EMT-associated proteins (E-cadherin, N-cadherin), and glycolysis-associated proteins (GLUT1, LDHA, and PDK1). Cellular metabolism assays detected changes in glucose metabolites. A xenograft model was constructed, and the mouse tumor weight and volumes were measured periodically. Dual-luciferase assays and ChIP assays were performed to authenticate the transcriptional regulation of TAL1 on PKM2 and the relationship between DNMT3B and TAL1.</p> Results <p>TAL1 was lowly expressed in NSCLC, and TAL1 overexpression prevented the proliferation, migration, and invasion and elevated apoptosis. TAL1 inhibited PKM2 transcription, and overexpression of PKM2 reversed the trend of overexpression of TAL1 and promoted glycolysis. DNMT3B inhibited TAL1 expression through methylation modification. DNMT3B overexpression facilitated NSCLC cell growth and promoted glycolysis, and further overexpression of TAL1 reversed this trend. In vivo experiments showed that overexpression of TAL1 inhibited NSCLC progression, while combined overexpression of PKM2 promoted NSCLC progression. Overexpression of DNMT3B promoted NSCLC progression, and combined knockdown of PKM2 inhibited NSCLC progression.</p> Conclusion <p>DNMT3B activates glycolysis and promotes NSCLC progression by mediating methylation modification of TAL1 and inducing PKM2 transcription.</p>