Moutan Cortex (MCE) has been used in traditional medicine to remove heat from the blood, promote blood circulation and alleviate blood stasis. This study was conducted to evaluate the effects of MCE on regulatory mechanisms of cytokines and nitric oxide (NO) involved in immunological activity of Raw264.7 cells. Cells were pretreated with methanolic extracts of MCE, and further cultured for an appropriate time after lipopolyssacharide (LPS) addition. During the entire experimental period, 0.1 and 0.3 mg ml−1 of MCE had no cytotoxicity. In these concentrations, MCE inhibited the production of NO and prostaglandin E2 (PGE2), the expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) and phosphorylated inhibitor of κBα (p-IκBα), and the activation of nuclear factor κB (NF-κB). MCE also reduced the concentration of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in the Raw264.7 cells that were activated by LPS. These results demonstrate that MCE has anti-inflammatory effects through the inhibition of iNOS and COX-2 expression by suppressing the phosphorylation of I-κBα and the activation of NF-κB.