Cardiovascular diseases are closely associated with dysfunction of vascular endothelial cells (VECs), which can be influenced by various intrinsic and extrinsic factors, including fibroblast growth factor 21 (FGF21), but the effects of serum FGF21 on VECs remain unclear. We performed a cross-sectional study nested within a pro-spective cohort to assess the range of physiological concentrations of fasting serum FGF21 in 212 healthy in-dividuals. We also treated human umbilical VECs (HUVECs) with recombinant FGF21 at different concentrations. The effects of FGF21 treatment on glycolysis, nitric oxide release and reduction of intracellular reactive oxygen species were assessed. The cells were also collected for RNA transcriptomic sequencing to investigate the po-tential mechanisms induced by FGF21 treatment. In addition, the roles of SIRT1 in the regulation of FGF21 were evaluated by SIRT1 knockdown. The results showed that the serum FGF21 concentration in healthy individuals ranged from 15.70 to 499.96 pg/mL and was positively correlated with age and pulse wave velocity. FGF21 at 400 pg/mL was sufficient to enhance glycolysis, increase nitric oxide release and protect cells from H2O2-induced oxidative damage. The upregulated genes after FGF21 treatment were mostly enriched in metabolic pathways, whereas the downregulated genes were mostly enriched in inflammation and apoptosis signaling pathways. Moreover, SIRT1 may be involved in the regulation of some genes by FGF21. In conclusion, our data indicate that FGF21 at a level within the physiological concentration range has a beneficial effect on HUVECs and that this effect may partly depend on the regulation of SIRT1.