A large number of macrophages in inflamed sites not only amplify the severity of inflammatory responses but also contribute to the deleterious progression of many chronic inflammatory diseases, autoimmune diseases and cancers. Macrophage migration is a prerequisite for their entry into inflammatory sites and their participation of macrophages in the pathologic processes. Inhibition of macrophage migration is therefore a potential anti-inflammatory mechanism. Moreover, alleviation of inflammation also prevents the macrophages infiltration. Sinomenine (SIN) is an alkaloid derived from the Chinese medicinal plant Sinomenium acutum. It has multiple pharmacological effects, including anti-inflammation, immunosuppression, and anti-arthritis. However, its anti-inflammatory molecular mechanisms and effect on macrophage migration are not fully understood. The purpose of this research was to investigate the pharmacological effects and the molecular mechanism of SIN on macrophage migration in vivo and in vitro as well as to elucidate its anti-inflammatory mechanisms associated with macrophage migration. Our results showed that SIN reduced the number of RAW264.7 cells migrating into inflammatory paws and blocked lipopolysaccharide (LPS)-induced RAW264.7 cells and bone marrow-derived macrophages (BMDMs) migration in vitro. Furthermore, SIN attenuated the 3D mesenchymal migration of BMDMs. The absence of macrophage migration after circulatory and periphery macrophages depletion led to a reduction in the severity of inflammatory response. In macrophages depleted (macrophages-/-) mice, as inflammatory severity decreased, RAW264.7 cells migration was suppressed. A non-obvious effect of SIN on the inflammatory response was found in macrophages-/- mice, while the inhibitory effect of SIN on RAW264.7 cells migration was still observed. Furthermore, the migration of RAW264.7 cells pre-treated with SIN was suppressed in normal mice. Finally, Src/focal adhesion kinase (FAK)/P130Cas axis activation, which supports macrophages mesenchymal migration, and iNOS expression, NO production, integrin αV and in integrin β3 expressions, which promote Src/FAK/P130Cas activation, were down-regulated by SIN. However, SIN had no obvious effect on the expression of the monocyte chemoattractant protein-1 (MCP-1), which is an important chemokine for macrophage migration. These results indicated that SIN significantly inhibited macrophage mesenchymal migration by down-regulating on Src/FAK/P130Cas axis activation. There was a mutual regulatory correlation between the inflammatory response and macrophage migration, and the effects of SIN on macrophage migration were involved in its anti-inflammatory activity. © 2021 Elsevier Ltd