BACKGROUND Non-small cell lung cancer (NSCLC) accounts for about 85% of all types of lung cancer. Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) is involved in DNA methylation, and DNA methylation is related to tumorigenesis. The role of MTHFD1 in NSCLC was examined in our study. MATERIAL AND METHODS The correlation between the expression of MTHFD1 and the clinicopathological features of patients diagnosed with lung cancer was investigated using the chi-square test. The viability and apoptosis of NCI-H1299 cells was respectively detected using cell counting kit-8 and flow cytometry assays. The expression levels of MTHFD1, apoptosis-related factors and DNA methyltransferase-related factors were assessed by quantitative real-time PCR (qRT-PCR) and western blot assays. RESULTS We found that MTHFD1 expression in the tumor tissues and cells was higher than that of adjacent normal tissues and cells. The survival time of patients with high MTHFD1 expression was shorter than those with low MTHFD1 expression. The expression level of MTHFD1 was related to tumor size, TNM stage, histologic grade, and metastasis, but not linked to gender and age. Besides, si-MTHFD1 significantly decreased the viability of cells in a time-dependent manner, and increased cell apoptosis. When cells were transfected with MTHFD1-siRNA, the levels of surviving and B-cell lymphoma-2 (Bcl-2) were attenuated, while p53 and Bcl-2 associated X protein (Bax) levels were enhanced. Moreover, si-MTHFD1 markedly downregulated the expression levels of DNA methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b. CONCLUSIONS Collectively, our results proved that MTHFD1 silencing obviously reduced the proliferation and enhanced the apoptosis of NSCLC via suppressing DNA methylation.