〔Abstract〕 Objective To investigate the expression of alpha-actinin-4(ACTN4) and the effect on cell proliferation in esophageal squamous cell carcinoma(ESCC). Methods The expression of ACTN4 in ESCC tissues and paired normal tissues was detected by immunohistochemistry, and the correlation between ACTN4 and the clinicopathologi-cal features was analyzed statistically. The ACTN4 shRNA lentiviral plasmids were constructed, and the stable ECA109 strain with ACTN4 shRNA knockdown was established using lentivirus packaging technology. The knockdown efficiency on protein level was checked by Western blot,and cell proliferation was detected by colony formation assays. The downstream target proteins were validated in ESCC cell line ECA109 based on the previous proteomics analyses in melanoma cell line A375 with or without ACTN4 shRNA knockdown. Results The expression of ACTN4 in ESCC tissues was significantly higher than that of normal tissues. ACTN4 shRNA stable knockdown ECA109 cell strains were successfully constructed. The results of colony formation assays showed that ACTN4knockdown inhibited the cell proliferation and down-regulated NADH ∶ Ubiquinone oxidoreductase core subunit V1( NDUFV1) protein expression in ECA109 cells. Conclusion Upregulation of ACTN4 in ESCC cells promotes the cell proliferation and enhances the protein expression of NDUFV1.