The G protein-coupled receptor GPR18, engaged by pro-resolving and cannabinoid-related lipid ligands, plays a vascular bed-specific protective role in endothelial function. The aim of the present study was to establish the vasoreactivity and passive biomechanical properties of the thoracic aorta and femoral artery of adult GPR18 knockout compared with wildtype mice, using ex vivo myography, arterial morphology, and immunohistochemistry. The results revealed heightened receptor-independent contractility, loss of prostanoid-dependent contractile responses, altered vascular smooth muscle cell (VSMC) calcium handling, and an attenuated stress–tension relationship in the thoracic aorta of GPR18 knockout mice. This phenotype was almost entirely reversed in the femoral artery, with attenuated receptor-independent contractility, unchanged VSMC calcium handling, and a heightened stress–tension relationship in GPR18 knockout mice. These vascular bed-specific differences highlight the need to consider tissue context in the development of GPR18-based vasculoprotective therapies for cardiovascular disease.