CML66 is a novel, promising tumor antigen; however, its biological roles remain unclear. In present study, we applied a short hairpin RNA triggered RNA interfering to suppress CML66 expression in HeLa cervical carcinoma cells. Knockdown of CML66 inhibited proliferation, migration and invasion activities of HeLa cells in vitro. Meanwhile, in nude mice, CML66 silencing suppressed tumor growth and pulmonary metastasis with HeLa cells injected subcutaneously. Furthermore, using metastasis-related genes cDNA microarrays, we found 9 genes were significantly down-regulated after CML66 silencing, including cathepsin L, MMP15, uPAR, VEGF, COX-2, S100A4, MUC1, MDM2 and RAC1.These results imply that CML66 may play an oncogenic role in ways of favoring tumor cells proliferation, invasion and metastasis-associated with multiple pathways. Thus, CML66 might be a potential target for development of cancer therapy. © 2008 Elsevier Ireland Ltd. All rights reserved.