The roots ofPeucedanum praeruptorumDunn andAngelica decursivaFranchet et Savatier are designatedZenko, which is a crude drug defined by the Japanese Pharmacopoeia. This crude drug is used as an antitussive and an expectorant and is included in the Kampo formulaJinsoin, which improves cough, fever, and headache. Although the anti-inflammatory effects of this crude drug have been determined, the constituents responsible for this effect remain unknown. To investigate biologically active compounds, rat hepatocytes were used, which produce proinflammatory mediator nitric oxide (NO) in response to proinflammatory cytokine interleukin 1β (IL-1β). A methanol extract ofP. praeruptorumroots, which suppressed IL-1β-induced NO production, was fractionated into three crude fractions (ethyl acetate (EtOAc)-soluble,n-butanol-soluble, and water-soluble fractions) based on hydrophobicity. The EtOAc-soluble fraction markedly inhibited NO production. After this fraction was purified, three biologically active compounds were identified as praeruptorins A, B, and E, the contents of which were high. A comparison of their activities indicated that praeruptorin B exhibited the highest potency to inhibit NO production by decreasing inducible NO synthase expression and suppressed the expression of mRNAs encoding proinflammatory cytokines. Collectively, the three praeruptorins may primarily contribute to the anti-inflammatory effects ofP. praeruptorumroots.