Yu Ping Feng San (YPFS), a Chinese herbal decoction comprising Astragali Radix (AR; Huangqi), Atractylodis Macrocephalae Rhizoma (AMR; Baizhu), and Saposhnikoviae Radix (SR; Fangfeng), has been used clinically to treat inflammatory bowel diseases (IBD). Previously, we demonstrated a dual role of YPFS in regulating cytokine release in cultured macrophages. In this study, we elucidated the anti-inflammatory effect of YPFS that is mediated through modulating the expression of three key enzymes involved in IBD: inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and intestinal alkaline phosphatase (IALP). In a lipopolysaccharide (LPS)-induced chronic-inflammation model of cultured murine macrophages, YPFS treatment suppressed the activation of iNOS and COX-2 expression in a dose-dependent manner. Conversely, application of YPFS in cultured small intestinal enterocytes markedly induced the expression of IALP in a time-dependent manner, which might strengthen the intestinal detoxification system. A duality of YPFS in modulating the expression of iNOS and COX-2 was determined here. The expression of iNOS and COX-2 in macrophages was induced by YPFS, and this activation was partially blocked by the NF-κB-specific inhibitor BAY 11-7082, indicating a role of NF-κB signaling. These YPFS-induced changes in gene regulation strongly suggest that the anti-inflammatory effects of YPFS are mediated through the regulation of inflammatory enzymes.