Abstract Quantitative diffusion tensor imaging (DTI) offers a valuable tool to probe the microstructural changes in neural tissues in vivo, where absolute quantitation accuracy and reproducibility are essential. It has been long recognized that measurement of apparent diffusion coefficient (ADC) using DTI could be influenced by the presence of water molecules in cerebrovasculature. However, little is known about to what extent such blood signal affects DTI quantitation. In this study, we quantitatively examined the effect of cerebral hemodynamic change on DTI indices by using a standard multislice echo planar imaging (EPI) spin echo (SE) DTI acquisition protocol and a rat model of hypercapnia. In response to 5% CO2 challenge, mean, radial and axial diffusivities measured with diffusion factor (b-value) of b=1.0 ms/μm2 were found to increase in whole brain (1.52%±0.22%, 1.66%±0.16% and 1.35%±0.37%, respectively), gray matter (1.56%±0.23%, 1.63%±0.14% and 1.47%±0.45%, respectively) and white matter regions (1.45%±0.28%, 1.88%±0.33% and 1.10%±0.26%, respectively). Fractional anisotropy (FA) was found to decrease by 1.67%±0.38%, 1.91%±0.59% and 1.46%±0.30% in whole brain, gray matter and white matter regions, respectively. In addition, these diffusivity increases and FA decreases became more pronounced at a lower b-value (b=0.3 ms/μm2). The results indicated that in vivo DTI quantitation in brain can be contaminated by vascular factors on the order of few percentages. Consequently, alterations in cerebrovasculature and hemodynamics can affect the DTI quantitation and its efficacy in characterizing the neural tissue microstructures in normal and diseased states. Caution should be taken in designing and interpreting quantitative DTI studies as all DTI indices can be potentially confounded by physiologic conditions and by cerebrovascular and hemodynamic characteristics.