KNDC1 (kinase noncatalytic C-lobe domain containing 1), a brain-specific Ras guanine nucleotide exchange factor, controls the negative regulation of neuronal dendrite growth. However, the effect of KNDC1 on cellular senescence remains to be elucidated. The present study investigated the impact of KNDC1 knockdown on human endothelial cell senescence and the mechanisms underlying this effect. Human umbilical vein endothelial cells (HUVECs) cultured in vitro were used as a model of biological aging. Senescence‑associated β-galactosidase staining was used to detect cellular senescence and flow cytometry was employed to determine cell cycle progression. Quantitative polymerase chain reaction (qPCR) and western blot analysis were utilized to investigate mRNA transcription and protein expression. In the HUVECs, a senescence-like phenotypes developed with increasing passage number in vitro, which were associated with a progressive increase in the transcription and expression of KNDC1. KNDC1 knockdown promoted cell proliferation and partially reversed cellular senescence and cell cycle arrest in the G0/G1 phase in aging HUVECs. Investigations into the mechanism underlying this effect demonstrated that KNDC1 knockdown promoted HUVEC proliferation via the extracellular signal-regulated kinase signaling pathway and delayed HUVEC senescence by inhibiting the p53-p21-p16 transduction cascade. In addition, the promotion of the capillary tube network formation and the increased expression of endothelial nitric oxide synthase revealed that the activity and function of endothelial cells were enhanced. In conclusion, KNDC1 knockdown delayed endothelial cell senescence and promoted HUVEC activity and function. These results demonstrated that KNDC1 may be a novel therapeutic target for the development of agents to extend human life.