It has been demonstrated that the circadian clock system could be a potential factor involved in inflammation and the progression of atherosclerosis. A previous study has reported that cryptochrome 1 (CRY1), which is a core clock component, is associated with regulating proinflammation. However, whether CRY1 is involved in atherosclerosis is currently unknown. In the present study, we aimed to explore the role of CRY1 in regulating atherosclerosis in apolipoprotein E (ApoE)-deficient mice and the underlying molecular mechanism. We found that CRY1 mRNA expression was significantly decreased in atherosclerotic patients compared to the healthy subjects. Overexpression of CRY1 in the mouse model of atherosclerosis by adenovirus-mediated gene transfer significantly decreased the expression of proinflammatory factors including tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-6, and macrophage inflammatory protein-1α (MIP-1α). In addition, the adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin, were also downregulated by CRY1 overexpression. Furthermore, the plaque area of the aortic sinus and the concentrations of total cholesterol (TC), triglyceride (TG), and low density lipoprotein cholesterol (LDL-C) were also decreased in the atherosclerotic mice by CRY1 overexpression. Moreover, overexpression of CRY1 significantly decreased the protein levels of toll-like receptor (TLR) 2, TLR4 and phosphorylated p65 (p-p65). Additionally, the results of luciferase reporter assay exhibited that CRY1 overexpression was capable of inhibiting the activation of nuclear factor-kappa B (NF-κB). Taken together, our results suggest that overexpression of CYR1 relieves the development of atherosclerosis that may be associated with regulating the TLR/NF-κB pathway.