Six compounds were isolated from an ethanol extract of Swertia mussotii and identified as 2-phenylethyl-β-D-glucoside (1), amaroswerin (2), 1,3,7,8-tetrahydroxyxanthone (3), swertiamarine (4), 1,3,8-trihydroxy-5-methoxyxanthone (5) and methylswertianin (6). Compounds 1, 2 and 6 were isolated from S. mussotii for the first time. The anti-inflammatory activities of the compounds were evaluated by determining their effect on the production of NO by LPS-stimulated RAW264.7 cells. Amaroswerin was the most potent inhibitor of NO release, with an IC50 value of 5.42 μg/mL. Treatment with amaroswerin inhibited expression of iNOS at both protein and mRNA levels. Amaroswerin also dose-dependently suppressed production of TNF-α, IL-6 and IL-1β and reduced expression of mRNA for these LPS-stimulated pro-inflammatory mediators. Amaroswerin thus inhibits the expression of iNOS, TNF-α, IL-6 and IL-1β by downregulating transcription in LPS-induced RAW264.7 macrophage cells, indicating that amaroswerin may be a valuable therapeutic agent for the treatment of inflammatory diseases.