ERK5 regulates invasiveness of osteosarcoma by inducing MMP-9. The purpose of this study is to determine the role of ERK5 in cellular invasion of osteosarcoma (OS). The human OS cell line (MG63, SaOS, and U2OS) and primary OS cells were used for the study. The expression of ERK5 and MMP-9 in each cell was examined by western blot or RT-PCR. To evaluate the biological role of ERK5, proliferation assay (MTT) and invasion assay (BD Matrigel™) were performed after silencing ERK5 using siRNA. MMPs expressions were analyzed using RT-PCR and zymography after silencing ERK5. ERK5 was distinctly overexpressed in U2OS and primary OS cell. Both of them also expressed MMP-9, which was not shown in MG63 and SaOS in RT-PCR. ERK5 silencing did not suppress the proliferation of OS cells. However, ERK5 silencing significantly reduced the number of invading cells in invasion assay. The expression of MMP-9 was specifically reduced after silencing ERK5. The zymography showed that the enzyme activity of MMP-9 was also reduced after ERK5 suppression. The expression of ERK5 regulates the invasion of OS cells by inducing MMP-9 expression. Therefore, ERK5 may be a new therapeutic target in invasive OS expressing MMP-9.Copyright © 2011 Orthopaedic Research Society.