To gain a better understanding of the mechanisms that underpin aortic calcification, rodent models have been previously utilised. Regions of calcium and phosphate deposition are commonly visualised using labor-intensive two-dimensional histomorphometric techniques. In this study, we developed a novel micro-computed tomography (µCT) imaging protocol to quantify calcification in vascular tissues using high resolution three-dimensional (3D) reconstructions of aortae derived from the well-established Ecto-nucleotide pyrophosphatase/phosphodiesterase-1 knockout (Enpp1-/-) mouse model of medial aortic calcification. A dual-contrast method was employed for specimen preparation and the application of corn oil as a submersion medium for the samples during scanning, which allowed the definition and quantification of soft tissue. 3D µCT was utilised to produce reconstructions of calcified and non-calcified aortae. A highly accurate quantification of a standardized region of calcium deposition was undertaken on these reconstructions. An excellent correlation between images obtained from µCT and those obtained with Alizarin red staining, of whole aortae for calcium deposition, was observed. This imaging protocol provides a powerful tool for studying the development of aortic calcification and potential therapeutic approaches for clinical intervention.