Abstract: Objective To research the upstream regulation mechanism of miR-133 b in esophageal squamous cell carcinoma. Methods The upstream transcription factors involved in the regulation of miR-133 b expression were predicted by bioinformatics analysis. The effects of IRF4 overexpression plasmid on the expression of IRF4 and miR-133 b were verified by qRT-PCR and Western blotting. The mechanisms of IRF4 regulating miR-133 b expression were verified by chromatin immunoprecipitation(ChIP) and dual-luciferase reporter assay. Results ConTra V3 predicted that the transcription factor IRF4 could bind to miR-133 b. The transfection of IRF4 overexpression plasmid could significantly up-regulate the expression of IRF4 at mRNA(t=-18.950, P=0.010) and protein(t=-4.061, P=0.042) levels, and promote the expression of miR-133 b(t=-8.681, P=0.005). ChIP( primer 1: t=-17.391, P=0.003; primer 2: t=-14.421, P=0.004) and dual-luciferase reporter assay(t=-22.112, P=0.010) confirmed that the transcription factor IRF4 could directly target miR-133 b. Conclusion IRF4 can directly target miR-133 b, which is helpful to construct the signal regulatory network of miR-133 b and provide a new target for the diagnosis and treatment of esophageal squamous cell carcinoma.