Abstract Background: Previously we reported both Kupffer cells (KC), resident hepatic macrophages, and invariant natural killer T (iNKT) cells suppress liver injury in a mouse model of biliary obstruction. This study aims to clarify the interactions between these two cell populations during the course of cholestatic liver injury. Methods: Wild type (WT) and Ja18-/- (iNKT-deficient) female C57Bl/6 mice between 8 and 12 weeks of age were used for all experiments. Mice were injected with magnetic beads 3 days prior to sham- or bile duct ligation- (BDL) surgery to facilitate separation of KC from infiltrating mononuclear phagocytes. On day 3 post-surgery, animals were euthanized, and bead-containing KC were isolated and analyzed by flow cytometry. KC mRNA levels were analyzed using real-time quantitative PCR. Results: A shift in KC phenotype from CD11blo/Ly-6C- to CD11bhi/Ly-6C+ was more prominent in WT than Jα18-/- BDL mice. This shift was not seen in either sham groups. KC from WT BDL mice express higher mRNA levels of IL-1β, IL-6, IL-12, MCP-1, iNOS, and IL-10 compared to KC from Jα18-/- BDL mice. Conclusion: iNKT cell-activated KC express CD11bhi/Ly-6C+ and produce more pro- and anti-inflammatory cytokines in a mouse model of biliary obstruction.