Diffusion Tensor Imaging (DTI) offers a valuable in vivo tool to characterize water diffusion behavior in biological tissues, particularly brain tissues. The accuracy of DTI derived parameters can directly affect the interpretation of underlying microstructures, physiology or pathologies. It is anticipated that measurement of apparent diffusion coefficient (ADC) using DTI could be influenced and complicated by the presence of water molecules in brain vasculature. However, little is known about to what degree does blood signal from vasculature affect the diffusion quantitation. In this study, we examined the effects of hypercapnia on DTI quantification in rat brains using inhalation of 5% carbon dioxide (CO 2). It was found that statistically significant changes occurred in parametric DTI maps in response to cerebrovascular challenges, indicating that vascular factors could interfere with in vivo DTI characterization of neural tissues. Consequently, hemodynamic alterations can potentially affect the DTI quantitation and detection of tissue microstructures and pathological alterations. Therefore, cautions must be taken when interpreting DTI parameters in vivo.