The study aimed to evaluate whether the treatment of primary cultured human endothelial cells with native low-density lipoprotein (nLDL) could induce their senescence and to uncover some of the putative mechanisms involved. For this purpose, human umbilical vein endothelial cells (HUVECs) were subcultured and/or continuously cultured with nLDL (0, 2, 5, and 10 μg protein/mL), for up to 9 days. The results indicated that nLDL inhibited the proliferation of HUVECs by arresting the cell cycle at G1 phase. The G1-arrested cells showed increase in cytosolic senescence-associated-β-galactosidase (SA-β-Gal) activity, a biomarker of cellular senescence. The causative factor of the cellular senescence was nLDL itself and not oxidized LDL (oxLDL), since blocking LDL receptor (LDLR) with the anti-LDLR antibody opposed the nLDL-induced increase of SA-β-Gal activity and decrease of cellular proliferation. In addition, nLDL-induced cellular senescence by inhibiting the phosphorylation of pRb (G1 arrest) via p53 as well as p16 signal transduction pathways. G1 phase arrest of the senescent cells was not overcome by nLDL removal from the culture medium. Moreover, the nLDL-treated cells produced reactive oxygen species (ROS) dose- and time-dependently. These results suggested, for the first time, that long-term treatment of nLDL could induce the premature senescence of endothelial cells.