Our post-GWAS functional analysis revealed that cathepsin L (CTSL) is an upstream regulator of CUX1, and it induces p16INK4a-dependent and atherosclerosis-associated senescence by indirectly activating CUX1 transcription in a process that requires its proteolytic activity. This suggests an unidentified transcription regulator between CTSL and CUX1, and CTSL-mediated cleavage of this regulator could transcribe CUX1, inducing senescence. Here, in search of this transcriptional regulator, we discovered that Notch1 is a substrate of CTSL, and CTSL can proteolytically activate Notch1 in a ligand-independent fashion, liberating NICD. NICD, after complexing with RBPJ in the nuclei, induces CUX1/p16INK4a-dependent senescence. Consistently, an upregulation of both CTSL and NICD, along with elevated cellular senescence in the plaques isolated from patients with atherosclerosis, was observed. In addition, we showed that endothelial deletion of CUX1 in the atherosclerosis-prone ApoE-/- mice blocks high-fat diet-induced senescence throughout the entire plaques, and these ApoE-/- mice exhibit similar phenotypes as the atherosclerosis-prone models with CTSL and Notch1/RBPJ inactivation including attenuated atherosclerotic lesion, intact and well-organized elastin fibers, and reduced macrophage content of plaque. This further supports our findings that both CTSL and Notch1/RBPJ are upstream regulators of CUX1, regulating senescence. Thus, while our studies identify a non-canonical Notch1 pathway that can be activated by CTSL in a ligand-independent fashion to induce senescence, our findings also reveal a role of senescence in the development of atherosclerosis. This provides new insight into developing drugs aimed to target cellular senescence for atherosclerosis.