The aim of the present study was to investigate the effect of HIF1α on Foxp3 expression in CD4+CD25− T lymphocytes. CD4+CD25− T lymphocytes were sorted from PBMC using a CD4+CD25+ regulatory T cell isolation kit. Lentivirus containing lentiviral vector that overexpressed HIF1α (HIF-lenti) and those containing empty expression vector (control-lenti) were produced. Meanwhile, lentivirus that contained lentiviral vector that suppressed HIF1α expression (siHIF-lenti) and those containing control vector (sicontrol-lenti) were also generated. The sorted CD4+CD25− T lymphocytes were infected with HIF-lenti, control-lenti, siHIF-lenti, and sicontrol-lenti, respectively. Approximately 72 hr after transduction, real-time PCR and Western blot were carried out to analyze the RNA and protein expression level of HIF1α and Foxp3. CD4+CD25− T lymphocytes cultured under 21% O2, 5% CO2 (normoxia) and 1% O2, 5% CO2 (hypoxia) were used as control. Our results showed that overexpression of HIF1α increased both mRNA and protein expression of Foxp3 and, meanwhile, suppression of HIF1α expression by RNAi could reverse high Foxp3 expression in CD4+CD25− T lymphocytes caused by hypoxic culture. These results suggested that hypoxia could stimulate Foxp3 expression by increasing HIF1α expression in CD4+ T lymphocytes which may promote CD4+ T lymphocytes to convert to Treg.