Objective To explore the mechanism of microRNA (miRNA)-340-5p in regulating inhibitors of DNA binding/differentiation 3 (ID3) and its effect on the biological function of esophageal squamous cell carcinoma (ESCC) cells. Methods The effect of miRNA-340-5p on ID3 expression in human ESCC Eca109 cells was preliminarily confirmed using bioinformatics analysis, quantitative real-time polymerase chain reaction and Western blotting. The expression levels of miRNA-340-5p and ID3 were detected in 12 pairs of ESCC tissues and adjacent normal tissues. The regulatory effect of miRNA-340-5p on ID3 was verified by dual-luciferase reporter assay. The effects of miRNA-340-5p on the proliferation, migration and invasion of Eca109 cells were evaluated using cell counting kit 8, colony formation assay and Transwell assay. Results Eleven miRNAs that most likely to bind to ID3 gene were identified through bioinformatics analysis and experimental verification, of which miRNA-340-5p had the most significant effect on ID3 expression at the transcriptional and translation levels. Dual luciferase reporter assay confirmed that miRNA-340-5p could directly bind to ID3 gene. The expression of miRNA-340-5p was significantly lower and the expression of ID3 mRNA was significantly higher in the ESCC tissues compared with the adjacent normal tissues (both P<0.01), and the expression of miRNA-340-5p was negatively correlated with the expression of ID3 mRNA in the ESCC tissues (r=-0.71, P<0.01). Cell function experiments showed that miRNA-340-5p could inhibit the proliferation, migration and invasion of Eca109 cells (all P<0.01). Conclusion miRNA-340-5p can inhibit the proliferation, migration and invasion of ESCC cells by targeting ID3 gene.