The vessel caliber index (VCI), a magnetic resonance imaging biomarker of the average blood vessel diameter, is increasingly being used as a tool for assessing tumor angiogenesis and response to antiangiogenic therapy. However, although the VCI has been correlated with histological vessel diameters, good quantitative agreement with histology has been lacking. In addition, no VCI validation studies have been performed in vivo where the structural deformations frequently associated with histological tissue preparation are not present. This study employs intravital optical microscopy (IVM) measurements of cerebral blood vessel diameters in a mouse orthotopic glioma model to provide the first such in vivo validation. Two VCI correlation models, both a linear and a 3/2-power dependence on the DeltaR2*/DeltaR2 ratio, were compared with the IVM data. The linear VCI model, determined from steady-state susceptibility contrast (SSC) images, was found to be in excellent quantitative agreement with the intravitally determined VCI for separate tumor size matched groups of mice. In addition, preliminary data indicate that the VCI is independent of whether a dynamic susceptibility contrast or SSC measurement method is used.