Homeobox genes encode transcription factors that are essential for normal development and are often dysregulated in cancers. The molecular mechanisms that cause their misregulation in cancers are largely unknown. In this study, we investigate the mechanism by which the Six1 homeobox protein, which has a crucial role during development, is frequently deregulated in several poor outcome, aggressive, metastatic adult human cancers, including breast cancer, ovarian cancer, hepatocellular carcinoma and pediatric malignancies such as rhabdomyosarcoma and Wilms' tumor. Our results reveal that miRNA-185 translationally represses Six1 by binding to its 3'-untranslated region. Analyses of ovarian cancers, pediatric renal tumors and multiple breast cancer cell lines showed decreased miR-185 expression, paralleling an increase in Six1 levels. Further investigation revealed that miR-185 impedes anchorage-independent growth and cell migration, in addition to suppressing tumor growth in vivo, implicating it to be a potent tumor suppressor. Our results indicate that miR-185 mediates its tumor suppressor function by regulating cell-cycle proteins and Six1 transcriptional targets c-myc and cyclin A1. Furthermore, we show that miR-185 sensitizes Six1-overexpressing resistant cancer cells to apoptosis in general and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in particular. Together, our findings suggest that the altered expression of the novel tumor suppressor miR-185 may be one of the central events that leads to dysregulation of oncogenic protein Six1 in human cancers.