Objective To explore the effect of forkhead box O1 (FOXO1) gene on the expression and transcription of vascular endothelial growth factor-A (VEGF-A) through overexpressing or silencing the FOXO1 gene in vitro. Methods The human immortalized gingival keratinocyte (HIGK) was cultured and transfected with ON-TARGET plus SMART pool for human FOXO1 siRNA or ON-TARGET plus Non-targeting Control Pool (control siRNA), the fluorescence intensity of VEGF-A was measured by the immunofluorescence staining in vitro. Luciferase assay was used to test the VEGF-A luciferase activity by FOXO1 overexpression or FOXO1 silence. Results The VEGF-A protein levels in HIGK transfected with FOXO1 siRNA significantly reduced by 57% compared with HIGK without transfection (P<0.05). The luciferase reporter results showed that overexpression of FOXO1 resulted in a 2.1-fold increase in VEGF-A transcriptional activity (P<0.05), and FOXO1 silencing produced a 20%-43% decrease in VEGF-A transcriptional activity (P<0.05). Conclusion FOXO1 in HIGKs can regulate VEGF-A protein expression.