Matrine has been used in anti-inflammatory and anticancer therapies for a long time. However, the antimetastatic effect and molecular mechanism(s) of matrine on osteosarcoma are still unclear. Therefore, the aim of this study was to assess the effects of matrine and related mechanism(s) on osteosarcoma cells. In the study, we found that matrine inhibited the proliferation of osteosarcoma cells in vivo and in vitro and inhibited tumor cell metastasis in vitro at cytotoxic doses. Matrine also decreased the expression of the matrix metalloproteinases-2 and 9, decreased p50 and p65 nuclear translocation, and decreased the phosphorylated level of I-κ-B (IκB)-β. In addition, matrine reduced the phosphorylated levels of extracellular signal-regulated kinase 1/2 proteins, which regulate the invasion of poorly differentiated cancer cells. Finally, when U2OS cells were grown as xenografts in nude mice, intragastric administration of matrine induced a significant dose-dependent decrease in tumor growth. These results show the anticancer properties of matrine, which include the inhibition of invasion and proliferation of human osteosarcoma cells.