Aim Rats with adenine‐induced chronic renal failure (A‐CRF) develop a reduced rate of relaxation of the thoracic aorta. The aim of this study was to elucidate the mechanisms underlying this abnormality. Methods Male Sprague Dawley rats received either chow containing adenine or were pair‐fed with normal chow (controls). After 8–14 weeks, arterial function was analysed ex vivo using wire myography and the expression of proteins involved in vascular smooth muscle excitation–contraction coupling in the thoracic aorta was analysed. Results The rate of relaxation following washout of KCl was reduced in A‐CRF rats vs. controls in the thoracic aorta (P < 0.01), abdominal aorta (P < 0.05), and common carotid artery (P < 0.05), but not in the common femoral artery. Relaxation rates of thoracic aortas increased (P < 0.01), but were not normalized, in response to washout of KCl with Ca2+‐free buffer. Microarray and qRT‐PCR analyses of genes involved in excitation–contraction coupling identified 10 genes, which showed significantly altered expression in A‐CRF thoracic aortas. At the protein level, the α2 subunit of the Na,K‐ATPase (P < 0.001) and SERCA2 (P < 0.05) was significantly downregulated, whereas stromal interaction molecule 1 and calsequestrin‐1 and calsequestrin‐2 were significantly upregulated (P < 0.05). Conclusions Rats with A‐CRF show a marked alteration in relaxation of larger conduit arteries localized proximal to the common femoral artery. This abnormality may be caused by reduced cytosolic Ca2+ clearance in vascular smooth muscle cells secondary to dysregulation of proteins crucially involved in this process.