Objective: To investigate the expression level of miR-9 in esophageal cancer and its effect on the biological function of esophageal cancer cells. Methods: The expression levels of miR-9 and Golgi phosphoprotein 3 (GOLPH3) in esophageal cancer and its adjacent tissues were detected by real-time fluorescence quantitative PCR (qRT-PCR). The miR-9 mimics was transfected into esophageal cancer EC109 cells, and the expression level of miR-9 was detected by qRT-PCR. The effects of overexpression of miR-9 on the biological function of EC109 cells were determined by the MTT assay, plate colony formation assay, Transwell migration assay and flow cytometry. The wild and mutant GOLPH3 double luciferase reporter gene vectors were constructed, and luciferase activity was detected. The effects of overexpression of miR-9 on the expression levels of GOLPH3 mRNA and protein were detected by qRT-PCR and Western blot. Results: Compared with the adjacent tissues, the expression level of miR-9 in esophageal cancer tissues decreased significantly (P＜0.01), while that of GOLPH3 increased significantly (P＜0.01). Compared with the negative control group, the expression level of miR-9 in EC109 cells transfected with miR-9 mimics increased significantly (P＜0.01), and the proliferation and migration ability of the EC109 cells decreased obviously (P＜0.01). The cell cycle of the EC109 cells was blocked in G2/M phase (P＜0.01). The dual luciferase reporter assay, qRT-PCR and Western blot confirmed that miR-9 could bind with GOLPH3 specifically (P＜0.01), and mediate the degradation of GOLPH3 mRNA (P＜0.01), which led to the decrease of GOLPH3 protein expression level (P＜0.05). Conclusion MiR-9 is low expression in esophageal cancer, and may participate in the occurrence and development of esophageal cancer by regulating GOLPH3.