The aim of this study was to evaluate whether the protective effect of intermittent clamping and ischemic preconditioning is related to an improved hepatic microcirculation after ischemia/reperfusion injury. Male C57BL/6 mice were subjected to 75 or 120 min of hepatic ischemia and 1 or 3 hours of reperfusion. The effects of continuous ischemia, intermittent clamping, and ischemic preconditioning before prolonged ischemia on sinusoidal perfusion, leukocyte-endothelial interactions, and Kupffer cell phagocytic activity were analyzed by intravital fluorescence microscopy. Kupffer cell activation was measured by tissue levels of tumor necrosis factor (TNF)-alpha, and the integrity of sinusoidal endothelial cells and Kupffer cells were evaluated by electron microscopy. Continuous ischemia resulted in decreased sinusoidal perfusion rate and phagocytic activity of Kupffer cell, increased leukocyte-endothelial interactions and TNF-alpha levels. Both protective strategies improved sinusoidal perfusion, leukocyte-endothelial interactions and phagocytic activity of Kupffer cells after 75-minutes of ischemia, and intermittent clamping also after 120 minutes ischemia. TNF-alpha release was significantly reduced and sinusoidal wall integrity was preserved by both protective procedures. In conclusion, both strategies are protective against ischemia/reperfusion injury by maintaining hepatic microcirculation and decreasing Kupffer cell activation for clinically relevant ischemic periods, and intermittent clamping appears superior for prolonged ischemia.