We previously reported the identification of HRPAP20 (hormone-regulated proliferation-associated protein 20), a novel hormone-regulated, proliferation-associated protein. In tumor cell lines, constitutive HRPAP20 expression enhanced proliferation and suppressed apoptosis, characteristics frequently associated with malignant progression. Here, we report that highly invasive breast cancer cell lines and human breast tumor specimens express elevated HRPAP20, which in transfection experiments in MCF-7 and MDA-MB-231 cells, increased invasion. Results from mechanistic studies revealed that HRPAP20 bound to calmodulin (CaM) via a conserved CaM-binding motif. Transfection of MCF-7 breast cancer cells with HRPAP20 harboring a mutated CaM-binding motif (HRPAP20K73A) inhibited its interaction with CaM and failed to increase invasion. Other experiments revealed that transfection with HRPAP20, but not HRPAP20K73A, increased secretion of matrix metalloproteinase-9 (MMP-9). Moreover, knockdown of HRPAP20 with small interfering RNA in MCF-7/HRPAP20 transfectants and wild-type MDA-MB-231 cells reduced invasion and inhibited secretion of MMP-9. Together these observations suggest that HRPAP20 may be an important regulator of breast tumor cell invasion by a CaM-mediated mechanism that leads to increased MMP-9 secretion. We conclude that dysregulation of HRPAP20 expression in tumor cells may contribute to the observed phenotypic changes associated with breast cancer progression.