Kupffer cells are the first line of defense in the liver against pathogens, yet several microbes successfully target the liver, bypass immune surveillance, and effectively develop in this tissue. Our current, albeit poor, understanding of Kupffer cell-pathogen interactions has been largely achieved through the study of primary cells, requiring isolation from a large numbers of animals. To facilitate the study of Kupffer cell biology, an immortalized rat Kupffer cell line, RKC1, was developed. We performed a comparative global proteomic analysis of RKC1 and primary rat Kupffer cells (PRKC) to characterize their respective responses to lipopolysaccharide (LPS)-mediated immune stimulation. We identified patent differences in the proteomic response profile of RKC1 and PRKC to LPS. We observed that PRKC upregulated more immune function pathways and exhibited marked changes in cellular morphology following stimulation. We consequently analyzed the cytoskeletal signaling pathways of these cells in light of the fact that macrophages are known to induce cytoskeletal changes in response to pathogens. Our findings suggest that Kupffer cells respond differently to inflammatory stimulus than do monocyte-derived macrophages, and such data may provide insight into how pathogens, such as the malaria parasite, may have evolved mechanisms of liver entry through Kupffer cells without detection.