Austroinulin (AI) and 6-O-acetyl-austroinulin (6-OAAI) are natural diterpenoids isolated from Stevia rebaudiana with anti-inflammatory activity. However, the mechanisms underlying their anti-inflammatory effects are not well understood. The purpose of this study was to investigate the effect of AI and 6-OAAI on nitric oxide (NO) production and their molecular mechanism in LPS-stimulated RAW264.7 macrophages. We found that AI and 6-OAAI inhibit the production of NO, iNOS, and pro-inflammatory cytokines (TNF-alpha, IL-6, IL-1beta, and MCP-1) in LPS-stimulated RAW264.7 macrophages. In these same cells, AI and 6-OAAI also suppressed the phosphorylation of STAT1 and the production of interferon-beta (IFN-beta). Moreover, treatment with AI and 6-OAAI inhibited the activation of interferon regulatory factor-3 (IRF3) and NF-kappaB. Taken together, our results suggest that AI and 6-OAAI inhibit NO production and iNOS expression by blocking the activation of STAT1, IRF3, and NF-kappaB in LPS-stimulated RAW264.7 macrophages. CI - Copyright (c) 2013 Elsevier Ltd. All rights reserved.