The aim of the present study was to investigate whether an expression of vascular cell adhesion molecule-1 (VCAM-1) was upregulated in 3-week simulated microgravity rat cerebral and carotid arteries and whether impaired endothelium-dependent relaxation was concomitant with VCAM-1 expression. Male Sprague-Dawley rats were randomly divided into control (CON) and hindlimb unweighting (HU) groups. After 3 weeks, the expression of the VCAM-1 protein and the vasodilatation of the basilar artery and common carotid artery were determined. Immunohistochemical results revealed positive staining of VCAM-1 on endothelial cells in these arteries from HU compared with CON rats. Western blot analysis confirmed an upregulated expression of VCAM-1 protein in these arteries from HU rats. Acetylcholine induced concentration-dependent vasodilatation in all artery rings, but with significantly smaller amplitude in the basilar artery (P < 0.01) and the common carotid artery (P < 0.05) from HU than those from CON rats. The data suggested that the expression of VCAM-1 protein was upregulated in cerebral and common carotid arteries of simulated microgravity rats, and the upregulation of VCAM-1 may contribute to impaired endothelium-dependent relaxation in simulated microgravity rat vasculature.