The anti-inflammatory activity of handelin (1), a guaianolide dimer from Chrysanthemum boreale flowers, was evaluated in vivo, and the effects on mediators nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) and the nuclear factor-κB (NF-κB) and ERK/JNK signaling pathways were investigated in vitro. Compound 1 inhibited lipopolysaccharide (LPS)-induced production of NO and PGE2 in cultured mouse macrophage RAW 264.7 cells. The suppression of NO and PGE2 production by 1 was correlated with the downregulation of mRNA and protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Compound 1 also suppressed the induction of pro-inflammatory cytokines TNF-α and IL-1β in LPS-stimulated RAW 264.7 cells. To further clarify the transcriptional regulatory pathway in the expression of iNOS and COX-2 by 1, the role of NF-κB was determined in RAW 264.7 cells. Compound 1 inhibits the binding activity of NF-κB into the nuclear proteins. The transcriptional activity of NF-κB stimulated with LPS was also suppressed by 1, which coincided with the inhibition of IκB degradation. Compound 1 also suppressed the activation of mitogen-activated protein kinases, including ERK and JNK signaling. In addition, the LPS-stimulated upregulation of miRNA-155 expression was suppressed by 1. The oral administration of 1 inhibited acute inflammation in carrageenan-induced paw and 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ear edema models. The serum level of IL-1β was also inhibited by 1 in a carrageenan-induced paw edema model. These findings suggest that the suppression of NF-κB activation and pro-inflammatory cytokine production may be a plausible mechanism of action for the anti-inflammatory activity of handelin.