The early events in an intestinal ischemic episode have been difficult to evaluate. Using in vivo microscopy we have analyzed in real-time the effects of short (15 min) and long (40-50 min) ischemia with subsequent reperfusion (IR) on structure, integrity and function of the mouse jejunal mucosa, while monitoring blood flow by confocal microscopy. IR was imposed by deflation/inflation of a vascular occluder and blood flow was monitored and confirmed with scanning confocal imaging. After short ischemia, villus tip cells revealed a rapid increase (23%) in the intracellular NAD(P)H concentration (confocal autofluorescence microscopy), and the pH-sensitive probe BCECF showed a biphasic response of the intracellular pH (pHi), quickly alkalinizing from the resting value of 6.8±0.1 to 7.1±0.1 but then strongly acidifying to 6.3±0.1. Upon reperfusion, values returned towards control. In contrast, results were heterogeneous after long ischemia and subsequent reperfusion. During long ischemia one-third of the epithelial cells remained viable with reversible changes upon reperfusion, but remaining cells lost membrane integrity (Lucifer Yellow, LY uptake) and had membrane blebs during ischemia. These effects became more pronounced as reperfusion progressed, when cells exhibited more severely affected NAD(P)H and pHi values, larger blebs, more LY uptake, and eventually cells were shed from the villus. These irreversible effects of IR occurred most prominently at the anti-mesenteric side of the intestine, most likely due to incomplete restoration of blood flow upon reperfusion in this region (stereo microscopy). We conclude that the adverse effects of short ischemia on the jejunum epithelium are fully reversible during reperfusion. However, after long ischemia, reperfusion cannot restore normal structure and functioning of a majority of cells, which deteriorate further. Our results provide a basis for defining the cellular events that cause tissue to transit from reversible to irreversible damage during IR.