Background: Sigesbeckia glabrescens Makino (SG) is one of the important plant origins of Sigesbeckiae herba and has been widely used for the treatment of chronic infammatory diseases in China. However, the underlying anti-infammatory mechanism of SG is rarely investigated and reported. There are more than 40 kinds of chemical constituents in SG, but the action of the bioactive compounds of SG is still unclear. Therefore, we aimed to systemically investigate the mechanisms behind the anti-infammatory properties of SG by combining in vitro and in silico investigations. Methods: Cytotoxicity was measured using the 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Nitric oxide (NO) release was detected using the Griess assay. The secretion of pro-infammatory cytokines and the expression of relevant proteins were assessed using ELISA kits and Western blots, respectively. Molecular docking was performed and scored using AutoDock via a comparison with the molecular docking of N-acetyl-d-glucosamine (NAG). Results: In lipopolysaccharides (LPS)-stimulated macrophages, SG signifcantly inhibited NO, MCP-1, and IL-6 secretion; iNOS expression; and NF-κB activation but did not signifcantly afect MAPK signalling (p38, ERK, and JNK). Moreover, the results from the molecular docking prediction suggested that over 10 compounds in SG could likely target TLR4, p105, and p65. Conclusions: These fndings suggest that the anti-infammatory efects of SG are highly related to the inactivation of NF-κB. Moreover, this study provides a novel approach to investigate the efects of herbal medicine using combined in vitro and in silico investigations.