〔Abstract〕 Objective To study the effect and mechanism of N6-methyladenine (m⁶ A) methylation modification enzyme YTH domain family protein 3 (YTHDF3) on the proliferation and migration of Ec109 esophageal squamous cell carcinoma cells. Methods Patients with esophageal cancer were selected as the research objects, and the expression of YTHDF3 gene mRNA and protein was detected in tumor tissues and adjacent tissues. pcDNA-YTHDF3, pcDNA, si-YTHDF3 and si-NC were transfected into Ec109 cells respectively. The mRNA expressions of YTHDF3, large tumor suppressor gene 1(LAST1), ankyrin repeat domain protein 1 (ANKRD1), cysteine-rich angiogenesis-inducible factor 61 (CYR61) and connective tissue growth factor (CTGF) were detected by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR); the protein expression of YTHDF3 gene was detected by Western blot; the cell proliferation activity was detected by MTT method. Transwell was used to detect cell migration. The relationship between YTHDF3 and survival was compared by Kaplan-Meier method. Results After transfection with si-YTHDF3, the mRNA and protein expressions of YTHDF3 and LAST1 genes, the mRNA expressions of target genes ANKRD1, CYR61 and CTGF in Ec109 cells were significantly decreased, and the cell proliferation and migration were significantly increased,the differences were statistically significant (P < 0.05). After transfection with pcDNA-YTHDF3, the mRNA and protein expressions of YTHDF3 and LAST1 genes, the mRNA expressions of target genes ANKRD1, CYR61 and CTGF in Ec109 cells were significantly increased, and the cell proliferation and migration were significantly decreased, the differences were statistically significant (P < 0.05). The expression of YTHDF3 gene mRNA and protein in tumor tissues of patients with esophageal squamous cell carcinoma was significantly lower than that in adjacent tissues, the differences were statistically significant (P < 0.05). The median survival time of patients with high expression of YTHDF3 gene was significantly higher than that of patients with low expression of YTHDF3 gene, the differences were statistically significant (P < 0.05). Conclusion YTHDF3 gene can inhibit the proliferation and migration of esophageal squamous cell carcinoma cells, and the mechanism may be related to the regulation of LAST1 gene expression.