〔Abstract〕 Objective To explore the effect of transcription factor ZNF174 on the stemness of non-small cell lung cancer(NSCLC). Methods StemChecker online analysis was employed to assess the impact of ZNF174 on tumor stemness markers. The ZNF174 knockdown plasmid was constructed via restriction enzyme digestion and enzyme-linked assembly, while the ZNF174 overexpression plasmid was generated through homologous recombination. Lentiviral infection established stable cell lines with ZNF174 knockdown or overexpression. Tumor stem cell spheroid formation assays were conducted to evaluate stemness maintenance capacity. RNA-seq and quantitative real-time polymerase chain reaction(qPCR) were utilized to detect effects on key stemness gene expression. Results The transcription factor ZNF174 promoted cellular stemness in non-small cell lung cancer(NSCLC). It promoted the expression of key stemness genes: B-cell-specific moloney murine leukemia virus insertion site 1(BMI1), acetaldehyde dehydrogenase 2(ALDH2), SRY-box transcription factor 2(SOX2), recombinant high mobility group AT hook protein 2(HMGA2), and snail family transcriptional repressor 1(Snail1) and enhanced tumor spheroid formation in NSCLC stem cells. Conclusion ZNF174 promotes the expression of tumor stemness genes in NSCLC and may become a novel therapeutic and prognostic target for NSCLC.