Background: Polyphyllin VII (PP7), a steroidal saponin from Paris polyphylla has been found to exert strong anticancer activity. Little is known about the anti-inflammatory property of PP7. In this study, the anti-inflammatory activity and its underlying mechanisms of PP7 were evaluated in LPS-stimulated RAW264.7 cells and in multiple animal models. Methods: The content of NO was determined by spectrophotometry. The levels of PGE2 and cytokines were measured by ELISA assay. The mRNA expression of pro-inflammatory genes was determined by qPCR. The total and phosphorylated protein levels were examined by Western blotting. The in vivo anti-inflammatory activities were evaluated by using mouse and zebrafish models. Results: PP7 reduced the production of NO and PGE2 and the protein and mRNA expressions of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and genes (iNOS, COX-2, and MMP-9) in LPS-induced RAW264.7 cells by suppressing the NF-κB and MAPKs pathways. Notably, PP7 markedly inhibited xylene-induced ear edema and cotton pellet-induced granuloma formation in mice, and suppressed LPS and CuSO4-induced inflammation and toxicity in zebrafish embryos. Conclusion: This study demonstrates that PP7 exerts strong anti-inflammatory activities in multiple in vitro and in vivo models and suggests that PP7 is a potential novel therapeutic agent for inflammatory diseases.