Vascular aging is an independent risk factor for age-related diseases, including atherosclerosis. Fibroblast growth factor 21 (FGF21) has been widely recognized as a metabolic regulator that is elevated in response to caloric and nutritional restrictions. Recent studies have demonstrated its emerging role as a pro-longevity hormone, but its effects on the senescence of human umbilical vascular endothelial cells (HUVECs) remain unclear. In the present study, we explored the anti-senescence effects and underlying mechanism of FGF21 on HUVECs. Co-cultivation of HUVECs with 5 ng/mL FGF21 significantly attenuated the phenotype changes of cells during in vitro subculture, including increased senescent population, decreased proliferation rate, decreased SIRT1 and elevated P53 and P21 protein levels. FGF21 also protected HUVECs from H2O2-induced cell damage, including premature cell senescence, intracellular accumulation of reactive oxygen species, increased DNA damage, decreased SIRT1 protein level and elevated protein levels of VCAM-1, ICAM-1, P53 and P21. Transient knockdown of SIRT1 in HUVECs significantly suppressed the protective effects of FGF21 for the rescue of H2O2-induced premature senescence and DNA damage, which suggests that the anti-senescence effect of FGF21 on HUVECs is SIRT1-dependent. These results support the potential of FGF21 as a therapeutic target for postponing vascular aging and preventing age-related vascular diseases.