Osteosarcoma is the most common primary malignancy of the bone. Aminopeptidase N (APN/CD13), a Zn+2-dependent ectopeptidase localized on the cell surface, is widely considered to influence the invasion mechanism. This study explores the potential involvement of APN in migration and invasion of human osteosarcoma cells in vitro using inhi-bitors and activators of APN. Cells treated with APN inhibitor bestatin displayed decreased migration and invasion in a Boyden chamber Transwell assay. Western blotting revealed reduced levels of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathway proteins, reduced phosphorylation of p38, ERK1/2 and JNK and decreased levels of NF-κB. Bestatin treatment also lowered APN, matrix metalloproteinase (MMP)-2 and -9 enzymatic activity and their mRNA expression. Reduced MMP-2 and -9 protein levels were also observed. By comparison, cells treated with cytokine interleukin-6 (IL-6), a stimulator of APN, displayed increased migration and invasion. Western blotting revealed increased levels of MAPK and PI3K pathway proteins, phosphorylated p38, ERK1/2 and JNK, and NF-κB. IL-6 treatment also increased APN and MMP-2 and -9 enzymatic activity. An increase of APN, MMP-2 and -9 mRNA levels, and MMP-2 and -9 protein levels was also observed. Together these experiments reveal potential enzymatic and signalling roles for APN in osteosarcoma and establish a starting point for an in-depth analysis of the role of APN in regulating invasiveness. A deeper knowledge about the regulatory mechanisms of APN may contribute to the development of anti-metastatic therapies.