Objective To investigate the effect of hypoxia on p53RFP gene expression and p53RFP promoter activity, and explore whether hypoxia responsive element (HRE) is involved in the regulation of p53RFP gene promoter activity by hypoxia. Methods HEK293 cells were cultured under normoxia and hypoxia conditions, p53RFP expressions at mRNA and protein levels were detected by real-time PCR and Western blot respectively. The HRE mutant p53RFP promoter dual luciferase reporter gene vector was constructed by site-directed mutagenesis technology and transfected into HEK293 cells under normoxia and hypoxia conditions. The promoter activities were detected by dual luciferase reporter system. Results Compared with the normoxia group, the expression of p53RFP mRNA in the hypoxia group increased significantly at different time points, and the difference was statistically significant(F=96.493,P<0.001); the expression of p53RFP and HIF-1α protein in the hypoxia group increased in a time-dependent manner. The luciferase reporter vectors containing p53RFP with mutant HRE were successfully constructed. The dual luciferase reporter gene assay showed that the activity of the wild-type p53RFP gene promoter was significantly increased under hypoxic conditions compared to normoxic condition(t=-19.504,P<0.001),and the promoter activity of p53RFP with HRE single mutation or double mutation were both significantly lower than that of wild type under hypoxic condition (F=160.891,P<0.001). Conclusions p53RFP gene expression was induced by hypoxia; the p53RFP promoter with mutant HRE dual luciferase reporter gene vectors were successfully constructed, and the HRE locus may play a key role in the hypoxia regulation of p53RFP gene promoter activity.