Diffuse reflectance spectroscopy (DRS) has been extensively used for characterization of biological tissues as a noninvasive optical technique to evaluate the optical properties of tissue. We investigated a method for evaluating the reduced scattering coefficient , the absorption coefficient , the tissue oxygen saturation , and the reduction of heme aa3 in cytochrome c oxidase CcO of in vivo liver tissue using a single-reflectance fiber probe with two source-collector geometries. We performed in vivo recordings of diffuse reflectance spectra for exposed rat liver during the ischemia–reperfusion induced by the hepatic portal (hepatic artery, portal vein, and bile duct) occlusion. The time courses of at 500, 530, 570, and 584 nm indicated the hemodynamic change in liver tissue as well as . Significant increase in during ischemia and after euthanasia induced by nitrogen breathing was observed, which indicates the reduction of heme aa3, representing a sign of mitochondrial energy failure. The time courses of at 500, 530, 570, and 584 nm were well correlated with those of , which also reflect the scattering by red blood cells. On the other hand, at 700 and 800 nm, a temporary increase in and an irreversible decrease in were observed during ischemia–reperfusion and after euthanasia induced by nitrogen breathing, respectively. The change in in the near-infrared wavelength region during ischemia is indicative of the morphological changes in the cellular and subcellular structures induced by the ischemia, whereas that after euthanasia implies the hepatocyte vacuolation. The results of the present study indicate the potential application of the current DRS system for evaluating the pathophysiological conditions of in vivo liver tissue.