Bilirubin is an endogenous substance derived from heme catabolism. In this study, we aimed to assess the anti-inflammatory activity of bilirubin, and to determine the mechanism thereof. The anti-inflammatory activity of bilirubin was evaluated using lipopolysaccharide (LPS)-treated peritoneal macrophages (PMs) and Raw264.7 cells, and mice with alum-induced peritonitis. The mRNA and proteins of NOD-like receptor family pyrin domain containing 3 (Nlrp3) and inflammatory cytokines were determined using qPCR and Western blotting, respectively. Distribution of phosphorylated (p) p65 [a NF-κB (nuclear factor-κB) subunit] in the cytoplasm and nucleus were evaluated by immunofluorescence analysis and electrophoretic mobility shift assay. Bilirubin prior to LPS treatment decreased protein expressions of Nlrp3, pro-interleukin (IL)-1β and mature IL-1β in PMs, whereas bilirubin post LPS treatment showed no effects. Bilirubin prior to LPS treatment dose-dependently repressed expressions of Nlrp3 and IL-1β, and inhibited translocation of p-p65 to nucleus in Raw264.7 cells. Bilirubin treatment decreased myeloperoxidase activity and reduced the levels of inflammatory cytokines (i.e., IL-1β, TNFα and IL-6) in lavage fluid in mice with alum-induced peritonitis. This was accompanied by a lower mortality rate. In addition, the mRNAs of Nlrp3 and IL-1β in peritoneal exudates cells were decreased, and the levels of p-p65 and mature IL-1β proteins were reduced. In conclusion, bilirubin acted on inflammation and alleviated alum–induced peritonitis through inactivation of Nlrp3 inflammasome.