〔Abstract〕 Objective To investigate the effect of miR-25-3 p targeting NOTCH1 gene on invasion, migration and proliferation of esophageal squamous carcinoma cells, and to elucidate the mechanism of miR-25-3 p targeting the regulation of NOTCH1 gene in esophageal squamous cell carcinoma. Methods ECA109 cells were uniformly inoculated into culture dishes at 1 × 106/ml,and each group was given 3 replicate wells. The experiment was divided into two groups. Group 1: miR-25-3 p up-regulated( transfected with miR-25-3 p mimics),miR-25-3 p down-regulated( transfected with miR-25-3 p inhibitor) and NC group; Group 2: NOTCH1 up-regulated( transfected NOTCH1 mimics),NOTCH1 down-regulated( transfected with NOTCH1 inhibitor),and NC group. Real-time quantitative PCR was used to detect the expression levels of miR-25-3 p and NOTCH1 in each group,transwell chamber was used to detect cell invasion ability,CCK-8 was used to detect cell proliferation,bioinformatics analysis and luciferase activity assay were used to determine whether NOTCH1 was Target gene of miR-25-3 p. Results NOTCH1 is the target gene of miR-25-3 p. Results revealed that cell invasion and proliferation increased with the overexpression of miR-25-3 p by miRNA mimics and decreased with the suppression of NOTCH1. Conclusion miR-25-3 p promotes the invasion and proliferation of human ESCC cells by targeting NOTCH1,and miR-25-3 p is expected to become a new target for ESCC treatment.