Severe trauma may initiate a systemic inflammatory response, which in turn may result in remote organ injury. After limb ischemia/reperfusion (I/R), intravital fluorescence microscopy was applied to the livers of normotensive rats to investigate the initiation of remote injury to the liver. Additionally, we determined whether Kupffer cell activation and tumor necrosis factor-α (TNF-α) were involved, via perfusion deficits, in such injury. TNF-α, measured by immunoassay, peaked at 30 minutes of reperfusion, but returned to baseline within 60 minutes. Limb I/R resulted in significant increases to global hepatocellular injury measured by alanine transaminase (ALT) and lethal hepatocyte injury as seen with intravital fluorescence microscopy. Although the number of perfused sinusoids went unchanged, a significantly augmented perfusion heterogeneity was measured. After 1.5 hours of reperfusion, both TNF-α and Kupffer cells were shown to contribute to global hepatocellular injury (e.g., ALT). After 3 hours, TNF-α was no longer essential for this injury, suggesting that some other mechanism(s) activated Kupffer cells and initiated hepatocellular injury. Using propidium iodide and fluorescence microscopy, we found that both TNF-α and Kupffer cell activation were necessary to drive hepatocytes toward lethal injury. No additional benefits were observed with a combination of TNF-α inhibition and Kupffer cell suppression. These results not only implicate both Kupffer cells and TNF-α in the initiation of remote hepatic injury, but suggest that sinusoidal perfusion deficits are not essential for the initiation of such injury. Other mechanism(s) are likely involved in the pathogenesis of remote hepatic parenchymal injury.