Objective To investigate the correlation between DNA methyltransferase 3a (DNMT3a) expression and prognosis of lung squamous cell carcinoma (LSCC), as well as to elucidate the potential molecular mechanisms of DNMT3a in LSCC progression. Methods A retrospective analysis was conducted on a cohort of 47 LSCC patients who underwent surgery in the Department of Thoracic Surgery, the Second Affiliated Hospital of Air Force Medical University between May 2009 and January 2014. DNMT3a expression in LSCC tissues and paired adjacent non-cancerous tissues was assessed using immunohistochemical (IHC) staining. Patients were categorized into two groups based on the median IHC score of DNMT3a in LSCC tissues: high DNMT3a expression group (n=25) and low DNMT3a expression group (n=22). Prognostic correlation was analyzed in combination with clinicopathological data and public biological databases. To explore the molecular mechanisms of DNMT3a in LSCC progression, H1703 LSCC cell lines with overexpressed DNMT3a were established using a lentiviral infection method, with the creation of DNMT3a overexpression group and control group. Functional phenotype experiments were then conducted to test the differences in cell proliferation and migration between the two groups. DNMT3a overexpression tumor xenograft models were also established in nude mice, with the creation of DNMT3a overexpression group and control group (3 mice per group), to observe the growth of subcutaneous xenograft tumors. Western blotting analysis was employed to detect the expression of related proteins in the two groups of cells and subcutaneous xenograft tumors. Functional rescue experiments involved treating DNMT3a overexpression cells with c-Myc inhibitor (10058-F4) and assessing cell proliferation using EdU proliferation staining. Subsequently, DNMT3a overexpression cells were infected with RNAi-Zinc finger E-box binding homeobox 1 (ZEB1) lentivirus to knock down ZEB1 expression, and a Transwell migration assay was utilized to detect cell migration. Finally, DNMT3a overexpression group and control group were treated with DNMT specific inhibitor (SGI-1027), and the effects of DNMT3a inhibition on cell proliferation and migration were observed in both overexpression and control groups. Results IHC analysis revealed significantly higher DNMT3a level in LSCC tissues compared to adjacent non-cancerous tissues (P<0.0001). High DNMT3a expression was closely associated with N stage, clinical stage and tumor differentiation degree (P<0.05 or P<0.01), and it was identified as an independent risk factor for poor prognosis in LSCC patients (P<0.05). Functional phenotype experiments indicated that DNMT3a overexpression group exhibited significantly higher colony formation number, proportion of EdU-positive cells, wound healing migration rate, and Transwell cell migration number compared with control group (P<0.05). The volume and weight of subcutaneous xenograft tumors in DNMT3a overexpression group were significantly higher than those in control group (P<0.001). Western blotting showed that the protein expression levels of c-Myc and ZEB1 in DNMT3a overexpression group were significantly higher than those in control group. Functional rescue experiments demonstrated a significant reduction in the proportion of EdU-positive cells after 10058-F4 treatment in DNMT3a overexpression group (P<0.05). Knockdown of ZEB1 led to a significant decrease in the number of Transwell cell migration in DNMT3a overexpression group (P<0.05), with no significant change in DNMT3a protein expression. Additionally, inhibition of DNMT3a with SGI-1027 resulted in a significant decrease in colony formation number and migration rate in both DNMT3a overexpression group and control group (P<0.05). Conclusions High expression of DNMT3a is a significant independent risk factor for poor prognosis of LSCC patients. DNMT3a is likely to promote the proliferation of LSCC by upregulating c-Myc expression and to enhance the migration of LSCC by increasing ZEB1 expression.