The inhibitory activity of extract and compounds isolated from the roots of Cratoxylum formosum ssp. pruniflorum against nitric oxide (NO) was evaluated using RAW264.7 cells. Isolation of the CH2Cl2 extract of C. formosum ssp. pruniflorum roots afforded ten known xanthones including six tri-oxygenated xanthones (1-6) and four tetra-oxygenated xanthones (7-10), respectively. Compound 7 showed the highest inhibitory activity against NO release with an IC50 value of 3.9 μM, followed by compound 8 with an IC50 value of 4.3 μM, respectively. In order to understand the mechanism of this anti-inflammatory activity, the transcriptional level of 7 was found to down regulate mRNA expressions of iNOS and COX-2 in dose-dependent manners, whereas 8 inhibited only iNOS mRNA expression but did not affect on that of COX-2 gene. Xanthones might be the main anti-inflammatory components in C. formosum ssp. pruniflorum.