Aim To explore the role of silent information regulator 1(SIRT1)/forkhead transcription factor O1 (FOXO1) in H₂S antagonism of H₂O₂-induced endothelial cell senescence. Methods The positive cell rate was calculated by the number of blue-stained cells (senescent cells) observed under the light microscope by senescence-associated β-gal (SA-β-gal) staining. Western blot was used to detect the expression levels of P21, P53, plasminogen activator inhibitor-1 (PAI-1), FOXO1, acetylated FOXO1 (ac-FOXO1), manganese superoxide dismutase (MnSOD) and catalase proteins, and biotin-switch assay was used to measure the expression of S-sulfhydration of SIRT1, and ROS detection was used to quantitatively evaluate intracellular reactive oxygen species (ROS) level. Results Treatment with 100 μmol/L H₂O₂ significantly increased the SA-β-gal staining-positive cell rate and the expression of P21, P53 and PAI-1 proteins, suggesting that the senescent cell model was successfully established, whereas 100 μmol/L NaHS significantly antagonized this effect, and the number of SA-β-gal staining-positive cells decreased significantly (P＜0.01), and the expression of P21, P53 and PAI-1 proteins significantly reduced (P＜0.01). Compared with the control group, the expression of SIRT1, FOXO1, ac-FOXO1, MnSOD and catalase proteins in the H₂O₂ group significantly decreased (P＜0.05 or P＜0.01), the ac-FOXO1/FOXO1 ratio significantly increased (P＜0.01), and the ROS level significantly increased (P＜0.01). Compared with H₂O₂ group, the expression of SIRT1, S-sulfhydration of SIRT1, FOXO1, ac-FOXO1, MnSOD and catalase proteins in the NaHS+H₂O₂ group was significantly increased (P＜0.05 or P＜0.01), and the ac-FOXO1/FOXO1 ratio was significantly decreased (P＜0.01), while the ROS level was significantly reduced (P＜0.01). Conclusion H₂S can antagonize H₂O₂-induced senescence of HUVEC by a mechanism related to promoting SIRT1 sulfhydrylation and reducing FOXO1 acetylation.