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Recent studies have suggested that microgravity-induced arterial remodelling contributes to post-flight orthostatic intolerance and that multiple mechanisms are involved in arterial remodelling. However, the initial mechanism by which haemodynamic changes induce arterial remodelling is unknown. Focal adhesions (FAs) are dynamic protein complexes that have mechanotransduction properties. This study aimed to investigate the role of FAs in simulated-microgravity-induced basilar and femoral arterial remodelling. A 4-week hindlimb-unweighted (HU) rat model was used to simulate the effects of microgravity, and daily 1-hour intermittent artificial gravity (IAG) was used to prevent arterial remodelling. After 4-week HU, wall thickness, volume of smooth muscle cells (SMCs) and collagen content were increased in basilar artery but decreased in femoral artery (P < 0.05). Additionally, the expression of p-FAK Y397 and p-Src Y418 was increased and reduced in SMCs of basilar and femoral arteries, respectively, by HU (P < 0.05). The number of FAs was increased in basilar artery and reduced in femoral artery by HU (P < 0.05). Furthermore, daily 1-hour IAG prevented HU-induced differential structural adaptations and changes in FAs of basilar and femoral arteries. These results suggest that FAs may act as mechanosensors in arterial remodelling by initiating intracellular signal transduction in response to altered mechanical stress induced by microgravity.