Guaiac resin, extracted from the heartwood of Guaiacum officinale L. or G. sanctum L., is speculated to have anti-inflammatory effects. Lignans were purified from guaiac resin (also known as gum guaiacum) by monitoring the nitric oxide (NO) production in rat hepatocytes treated with an inflammatory cytokine interleukin-1β (IL-1β). Six lignans were purified from guaiac resin and identified as: dehydroguaiaretic acid (1), (+)-trans-1,2-dihydrodehydroguaiaretic acid (2), furoguaiaoxidin (3), meso-dihydroguaiaretic acid (4), furoguaiacin (i.e., α-guaiaconic acid) (5), and nectandrin B (6). To our knowledge, this is the first time that 1 has been isolated from guaiac resin as a non-derivative. Compounds 2 and 6 were first found in guaiac resin. Compound 3 was first isolated from a natural source as a non-derivative. Furthermore, 1-6 significantly suppressed NO production in IL-1β-treated hepatocytes. Because anti-inflammatory compounds suppress NO production, this system is often used to measure the anti-inflammatory effects of Kampo drugs and herbal constituents. The NO-suppressing activity of the six lignans isolated in this study indicates that guaiac resin has anti-inflammatory effects and that these lignans may be responsible for the anti-inflammatory effects of guaiac resin.