X-linked inhibitor of apoptosis protein (XIAP) overexpression has been found to be associated with malignant cancer progression and aggression in individuals with many types of cancers. However, the molecular basis of XIAP in the regulation of cancer cell biological behavior remains largely unknown. In this study, we found that a deficiency of XIAP expression in human cancer cells by either knock-out or knockdown leads to a marked reduction in -actin polymerization and cytoskeleton formation. Consistently, cell migration and invasion were also decreased in XIAP-deficient cells compared with parental wildtype cells. Subsequent studies demonstrated that the regulation of cell motility by XIAP depends on its interaction with the Rho GDP dissociation inhibitor (RhoGDI) via the XIAP RING domain. Furthermore, XIAP was found to negatively regulate RhoGDI SUMOylation, which might affect its activity in controlling cell motility. Collectively, our studies provide novel insights into the molecular mechanisms by which XIAP regulates cancer invasion and offer a further theoretical basis for setting XIAP as a potential prognostic marker and specific target for treatment of cancers with metastatic properties.The X-linked inhibitor of apoptosis protein (XIAP) 3 is a member of the IAP family that has received substantial attention during the last few years. Biochemical and structural studies have indicated that XIAP has three zinc-binding baculovirus IAP repeat (BIR) domains (BIR1-3), a loop region, and a RING finger (1). The BIR3 domain of XIAP is able to bind and inhibit caspase-9, whereas the BIR2 region binds and inhibits active caspase-3 and caspase-7. The RING domain of XIAP has E3 ligase activity and is able to degrade proteins by linking them to ubiquitin molecules (2-4). More recently, XIAP has been found to be a regulator of the cell cycle through binding the cell cycle regulators MAGED1 and NRAGE and to play an important role in the control of intracellular copper levels through ubiquitin ligase-dependent regulation of the copper-regulating gene MURR1 (5, 6). The ability of XIAP to regulate these pathways, uncoupled with its caspase inhibitory activities, indicates its distinct properties and functions. Based on the finding that XIAP-deficient mice do not display obvious apoptotic phenotypes (7), it was hypothesized that there might be new functions and signaling pathways affected by XIAP, which are probably distinct from those involved in apoptotic caspase cascades.There is growing evidence showing the correlation between high XIAP overexpression and malignant cancer aggression (8, 9). Comparison of XIAP expression between adjacent malignant tissue and normal tissue invariably demonstrates that XIAP is much more highly expressed in the malignant cancer tissue (10 -20). Poorly differentiated carcinomas also display significantly higher levels of XIAP expression than do well differentiated carcinomas (13,(17)(18)(19)(20). Moreover, XIAP expression in metastatic specimens is much higher than that in primary cancers ...