Although Kupffer cells (KCs) are capable of producing important growth‐stimulating cytokines, their role in liver regeneration following partial hepatectomy (PH) remains poorly understood. In the present study liver regeneration was studied after KC‐depletion by intravenous administration of liposome‐encapsulated dichloromethylene‐diphosphonate (Cl₂MDP), a method known to physically eliminate KCs. Furthermore, splenectomy was performed one week prior to PH to exclude the effect of Cl₂MDP‐liposomes on macrophage populations in the spleen. KC‐depletion was confirmed in cryostat liver sections stained with the monoclonal antibody ED2, a marker for resident tissue macrophages. Forty‐eight hours after PH, the cumulative hepatocyte DNA synthesis, as determined in liver sections by the hepatocyte bromodeoxyuridine labeling index, was significantly decreased in KC‐depleted rats when compared to control‐rats. The weight of the remnant liver, expressed as a percentage of the initial liver weight, was significantly less at 96 h after PH in KC‐depleted rats. KC‐depletion abolished the hepatic interleukin‐6 (IL‐6) and interleukin‐10 (IL‐10) mRNA synthesis and decreased hepatic expression of tumor necrosis factor‐α (TNF‐α), hepatocyte growth factor (HGF) and transforming growth factor‐β₁ (TGF‐β1) mRNA after PH, as was assessed by reverse‐transcriptase polymerase chain reaction (RT‐PCR). Moreover, at 4 h after PH the systemic release of IL‐6 was significantly decreased in KC‐depleted rats. We conclude that KCs are important for hepatocyte regeneration after PH. Delayed liver regeneration in KC‐depleted rats can be explained, at least in part, by an imbalanced hepatic cytokine expression, thereby suppressing important growth‐stimulating cytokines.