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Malignant fibrous histiocytoma (MFH), a high-grade, undifferentiated sarcoma, is highly aggressive, resistant to radiochemotherapy and associated with poor prognosis. There are no specific immunohistochemical markers for its diagnosis. The MFH cell line SFT7913 served as and immunogen for the generation of the FU3 monoclonal antibody in our laboratory. FU3 reacted strongly with MFH cells and with perivascular mesenchymal cells. In this study, we demonstrated that the antigen recognized by FU3 was identical to aminopeptidase N (APN/CD13) using FU3 immunoaffinity chromatography and N-terminal amino acid sequencing. Frequent (80%) and high-grade (>50% of cells) expression of APN/CD13 was observed in MFH, although low-grade expression was seen in some other sarcomas. Moreover, small interfering RNA (siRNA) that specifically targets APN/CD13 significantly suppressed MFH cell invasion in vitro. The newly developed monoclonal antibody FU3 specifically recognizes CD13 on MFH cells. Decreased expression of CD13, mediated by siRNA-mediated knockdown, attenuated the invasive capacity of MFH cells. Thus, results indicate that APN/CD13 could be an important diagnostic biomarker and therapeutic target for MFH.