This study investigates the effect of miR-141 overexpression on the proliferation and apoptosis of tongue squamous cell carcinoma (TSCC) cell lines (Tscca and Tca8113). TSCC cells were stably transfected with a miR-141 lentivirus (Ubi-miR-141-SV40-EGFP-IRES-puromycin, miR-141 group) or an empty vector (Ubi-MCS-SV40-EGFP-IRES-puromycin, miR-NC group), with untreated cells serving as the Blank group. miR-141 expression was evaluated by rt-qPCR, transfection efficiency and apoptosis were assessed by flow cytometry, and cell proliferation was measured using the CCK-8 assay. Western blot and rt-qPCR were used to analyze the protein and mRNA expression of phosphatase and tensin homolog (PTEN) and B-cell lymphoma-2 (BCL2). Results: Flow cytometry confirmed successful establishment of stably transfected cell lines. The CCK-8 assay showed no significant difference in proliferation between the miR-NC and Blank groups (P > 0.05), while proliferation was significantly reduced in the miR-141 group. Apoptosis was significantly higher in the miR-141 group compared to both the miR-NC and Blank groups. Western blot and rt-qPCR analyses revealed upregulation of pten gene and PTEN protein expression, as well as downregulation of bcl2 gene and BCL2 protein expression in the miR-141 group relative to the miR-NC and Blank groups. Conclusion: In TSCC cells with high miR-141 expression, pten gene and PTEN protein levels were elevated, while bcl2 gene and BCL2 protein levels were reduced. These findings indicate that miR-141 may regulate the expression of PTEN and BCL2 at both the gene and protein levels, thereby influencing cell proliferation, migration, and apoptosis. To our knowledge, this is the first study to explore the effects of miR-141 transfection in TSCC, providing valuable insights into its role in tumor progression and potential regulatory targets.