Arsenic trioxide inhibits osteosarcoma cell invasiveness via MAPK signaling pathway. Arsenic trioxide (As(2)O(3)) is an active ingredient in traditional Chinese medicine. Recent studies showed that it causes apoptosis in several cancer cells. However, research of As(2)O(3) in osteosarcoma is sparse. In our present study, an inhibitory effect of As(2)O(3) on osteosarcoma cell adhesion and metastasis was observed with a cell adhesion, migration and invasion test. The impact of As(2)O(3) on the activities of MMP-9 and MAPK pathway-related downstream factors was analyzed by western blotting. Our results showed that As(2)O(3) significantly inhibited motility, migration and invasion in HOS and MNNG cells in a concentration-dependent manner at concentrations ranging from 0.5-2 μM, and led to cytoskeletal rearrangements. As(2)O(3) exerted an inhibitory effect on the phosphorylation of ERK1/2 and MEK, which are the members of the MAPK family. Additionally, treatment with As(2)O(3) in combination with inhibitors specific for MEK (U0126) in HOS and MNNG cells resulted in a marked inhibition of cell invasion and As(2)O(3) could significantly reduce PMA-induced invasion. In conclusion, we demonstrate the inhibitory effects of As(2)O(3) on the invasiveness of HOS and MNNG cells, which may be due at least partly to inactivation of the MAPK signaling pathway.