Paeonia lactiflora Pall is one of the most well-known herbs in China, Korea, and Japan for more than 1200 years. Paeoniflorin, the major bioactive component of paeony root, has recently been reported to have anti-colitic activity. However, the underlying molecular mechanism is unclear. The present study was to explore the possible mechanism of Paeoniflorin in attenuating dextran sulfate sodium (DSS)-induced colitis. Pre- and co-administration of Paeoniflorin significantly reduced the severity of colitis and resulted in downregulation of several inflammatory parameters in the colon, including the activity of myeloperoxidase (MPO), the levels of TNF-α and IL-6, and the mRNA expression of pro-inflammatory mediators (MCP-1, Cox2, IFN-, TNF-α, IL-6 and IL-17). The decline in the activation of NF-B p65, ERK, JNK and p38 MAPK correlated with a decrease in mucosal Toll-like receptor 4 (TLR4) but not TLR2 or TLR5 expression. In accordance with the in vivo results, Paeoniflorin downregulated TLR4 expression, blocked nuclear translocation of NF-B p65, and reduced the production of IL-6 in LPS-stimulated mouse macrophage RAW264.7 cells. Transient transfection assay performed in LPS-stimulated human colon cancer HT-29 cells indicated that Paeoniflorin inhibits NF-B transcriptional activity in a dose-dependent manner. TLR4 knockdown and overexpression experiments demonstrated a requirement for TLR4 in Paeoniflorin-mediated downregulation of inflammatory cytokines. Thus, for the first time, the present study indicates that Paeoniflorin abrogates DSS-induced colitis via decreasing the expression of TLR4 and suppressing the activation of NF-B and MAPK pathways.