Timely evaluation of specific pathogenic mechanisms that underlie ischemia-reperfusion injury is essential to improve our understanding of the pathogenesis and to help develop effective therapies. Herein we report employment of intravital microscopy to visualize ischemia and reperfusion of rat liver in real time in vivo. We show that the time-lapse autofluorescence images exhibited a unique spatiotemporal change during the time course of ischemia and reperfusion. The intensity decreased rapidly after ischemia, but recovered gradually during reperfusion, with a rate depending strongly on the duration of ischemia. The intravital imaging further enabled direct visualization of occlusion of local microcirculation and showed that it led to delayed reoxygenation of cells, a pathogenic mechanism that would inevitably contribute tissue damage. Supported with complimentary hypoxia-reoxygenation experiments and inhibitory assays performed on cultured hepatocytes, we conclude that the variation of autofluorescence is mechanistically linked with the conversion of mitochondrial flavoproteins between the non-fluorescent reduced state and fluorescent oxidized state.