Kaempferol‑3‑O‑β‑rutinoside is one of the compounds isolated from tartary buckwheat (Fagopyrum tatricum), and its biological effects have not been studied yet. The present study examined the anti‑inflammatory effects of kaempferol‑3‑O‑β‑rutinoside and explore its regulatory mechanisms in lipopolysaccharide (LPS)‑induced macrophage RAW264.7 cells. Kaempferol‑3‑O‑β‑rutinoside exhibited no cytotoxic effect in RAW 264.7 macrophage and 293 cell lines up to 300 µM. As the concentration of kaempferol‑3‑O‑β‑rutinoside was increased, the activity of nitric oxide was inhibited in LPS‑stimulated RAW264.7 cells. In addition, kaempferol‑3‑O‑β‑rutinoside treatment downregulated the expression of inflammation‑related cytokines tumor necrosis factor‑α and interleukin‑6 in LPS‑stimulated RAW264.7 cells. Furthermore, kaempferol‑3‑O‑β‑rutinoside treatment suppressed inflammatory‑mediated factors, such as inducible nitric oxide synthase and cyclooxyganse‑2. These inflammation‑related proteins are known to be regulated by NF‑κB and mitogen‑activated protein kinase (MAPK) signaling, therefore the effect of kaempferol‑3‑O‑β‑rutinoside on these pathways was investigated. The results demonstrated that kaempferol‑3‑O‑β‑rutinoside decreased the expression of inhibitor of κB (IκB) protein and IκB kinases; as a result, the nuclear translocation and expression of NF‑κB was inhibited in LPS‑stimulated RAW264.7 cells. Furthermore, kaempferol‑3‑O‑β‑rutinoside inhibited the phosphorylation of p38, extracellular signal‑regulated kinase and stress‑activated protein kinase in LPS‑stimulated RAW264.7 cells. Thus, the present data demonstrated that kaempferol‑3‑O‑β‑rutinoside suppressed inflammation‑related gene expression through the NF‑κB and MAPK pathways, and suggested that it may be a useful reagent in pharmacological research.