Wilson disease (WD) is an autosomal recessive disorder of copper (Cu) metabolism. The gene responsible for WD, ATP7B, is involved in the cellular transport of Cu, and mutations in the ATP7B gene induce accumulation of Cu in the liver and ultimately in the brain. In a pilot study, the natural variations of copper stable isotope ratios (Cu-65/Cu-63) in the serum of WD patients have been shown to differ from that of healthy controls. In the present study, we challenged these first results by measuring the Cu-65/Cu-63 ratios in the blood of treated (n = 25), naive patients (n = 11) and age matched healthy controls (n = 75). The results show that naive patients and healthy controls exhibit undistinguishable Cu-65/Cu-63 ratios, implying that the Cu isotopic ratio cannot serve as a reliable diagnostic biomarker. The type of treatment (d-penicillamine vs. triethylenetetramine) does not affect the Cu-65/Cu-63 ratios in WD patients, which remain constant regardless of the type and duration of the treatment. In addition, the Cu-65/Cu-63 ratios do not vary in naive patients after the onset of the treatment. However, the Cu-65/Cu-63 ratios decrease with the degree of liver fibrosis and the gradient of the phenotypic presentation, i.e. presymptomatic, hepatic and neurologic. To get insights into the mechanisms at work, we study the effects of the progress of the WD on the organism by measuring the Cu concentrations and the Cu-65/Cu-63 ratios in the liver, feces and plasma of 12 and 45 week old Atp7b(-/-) mice. The evolution of the Cu-65/Cu-63 ratios is marked by a decrease in all tissues. The results show that Cu-63 accumulates in the liver preferentially to Cu-65 due to the preferential cellular entry of Cu-63 and the impairment of the Cu-63 exit by ceruloplasmin. The hepatic accumulation of monovalent Cu-63(+) is likely to fuel the production of free radicals, which is potentially an explanation of the pathogenicity of WD. Altogether, the results suggest that the blood Cu-65/Cu-63 ratio recapitulates WD progression and is a potential prognostic biomarker of WD.