Background Vascular endothelial cell (EC) alignment in the direction of flow is an adaptive response that protects against aortic diseases, such as atherosclerosis. The Rho GTPases are known to regulate this alignment. Herein, we analyze the effect of ARHGAP18 on the regulation of EC alignment and examine the effect of ARHGAP18 deficiency on the development of atherosclerosis in mice. Methods and Results We used in vitro analysis of ECs under flow conditions together with apolipoprotein E−/−Arhgap18−/− double-mutant mice to study the function of ARHGAP18 in a high-fat diet-induced model of atherosclerosis. Depletion of ARHGAP18 inhibited the alignment of ECs in the direction of flow and promoted inflammatory phenotype, as evidenced by disrupted junctions and increased expression of nuclear factor-κB and intercellular adhesion molecule-1 and decreased endothelial nitric oxide synthase. Mice with double deletion in ARHGAP18 and apolipoprotein E and fed a high-fat diet show early onset of atherosclerosis, with lesions developing in atheroprotective regions. Conclusions ARHGAP18 is a protective gene that maintains EC alignments in the direction of flow. Deletion of ARHGAP18 led to loss of ECability to align and promoted atherosclerosis development.