In an attempt to identify bioactive natural products with anti-inflammatory activity, we evaluated the anti-inflammatory potential of the indole-containing fraction from the roots of Brassica rapa (IBR) (Family Brassicaceae) and the underlying mechanisms. Initially, we examined the inhibitory effect of IBR on the production of pro-inflammatory mediators in vitro and then evaluated its in vivo anti-inflammatory effects. IBR was found to concentration-dependently reduce the productions of nitric oxide, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-induced macrophages. Consistent with these findings, IBR suppressed the LPS-induced expressions of inducible nitric oxide synthase (iNOS) at the protein level and of iNOS, TNF-α, and IL-6 at the mRNA level. Furthermore, IBR attenuated LPS-induced DNA-binding activities of nuclear factor-κB (NF-κB), and this was accompanied by a parallel reduction in the degradation and phosphorylation of inhibitory κBα and, consequently, by a reduction in the nuclear translocation of the p65 subunit of NF-κB. In addition, treatment with IBR inhibited carrageenan-induced paw edema in rats and acetic acid-induced writing response in mice. Taken together, our data suggest that the expressional inhibitions of iNOS, TNF-α, and IL-6 caused by an attenuation of NF-κB activation are responsible for the anti-inflammatory and antinociceptive activity of IBR.