The flowers of Hosta plantaginea (Lam.) Aschers are commonly used for the treatment of inflammatory diseases in traditional Chinese medicine with limited scientific evidence. Plantanone C (PC) is a new phytochemical isolated from H. plantaginea flowers; nevertheless, the anti-inflammatory effect remains unknown. Herein, we aimed to study the anti-inflammatory effects of PC and its underlying molecular mechanisms in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. The cell viability of PC-treated RAW 264.7 macrophage was measured by the Cell Counting kit-8 (CCK-8) assay. The anti-inflammatory effect of PC was investigated by measuring the levels of inflammatory mediators and pro-inflammatory cytokines using the Griess reaction and enzyme-linked immunosorbent assay (ELISA). Furthermore, the mechanism of action of PC was evaluated by Western blot analysis. The results showed that PC was not cytotoxic at concentrations as high as 40 μM. Furthermore, PC potently suppressed LPS-stimulated overproduction of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and IL-6 in RAW 264.7 macrophages. Western blot demonstrated that PC remarkably suppressed the phosphorylation of nuclear factor kappa-B (NF-κB) p65, inhibitor of NF-κB (IκB), c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinase (Erk), p38, and protein kinase B (Akt), as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in a concentration-dependent manner. Taken together, these findings suggest that PC exhibits anti-inflammatory effects by inhibiting NF-κB, iNOS, COX-2, mitogen-activated protein kinases (MAPKs), and Akt signaling pathways in RAW 264.7 macrophages.