Innate immune regulatory factor (IIRF) is a serum-derived biological response modifier, or agent that modifies/bolsters the pathogenic immune response. IIRF has been shown to provide protection (e.g., increase host survival) against a variety of pathogenic challenges including Salmonella typhimurium and Pasteurella multocida. In this report, we analyzed cytokine production in mice, whole blood and cultured cells in response to IIRF challenge. When IIRF was administered to mice, it stimulated the release of pro-inflammatory cytokines. Both IL-6 and IL-10 were temporally stimulated when IIRF was introduced. Serum IL-6 peaked at 3h (3957pg/ml) before returning to baseline by 8h, while IL-10 began to appear at 3h, peaked at 8h (734.5pg/ml), and returned to baseline by 36h. IIRF challenge also increased the expression of two positive acute phase proteins: haptoglobin increased 61-fold, while serum amyloid A levels increased 3500-fold in mice. A whole blood immunoassay indicated the effect of IIRF on production of IL-6 was dose- and time-dependent. Anti-asialo GM 1.1 provided to partially (>95%) eliminate functional natural killer cells elevated IL-6 in whole blood. IIRF was also able to induce the production of IL-6 in cultured human monocytic THP-1 cells. Based upon this study and previously reported data, we propose that IIRF activates the innate immune response via induction of IL-6 production.