MicroRNAs (miR) have been identified as posttranscriptional modifiers of target gene regulation and control the expression of gene products important in cancer progression. Here, we show that miR-661 inhibits the expression of metastatic tumor antigen 1 (MTA1), a widely up-regulated gene product in human cancer, by targeting the 3' untranslated region (UTR) of MTA1 mRNA. We found that endogenous miR-661 expression was positively regulated by the c/EBPalpha transcription factor, which is down-regulated during cancer progression. c/EBPalpha directly interacted with the miR-661 chromatin and bound to miR-661 putative promoter that contains a c/EBPalpha-consensus motif. In addition, we found that the level of MTA1 protein was progressively up-regulated, whereas that of miR-661 and its activator, c/EBPalpha, were down-regulated in a breast cancer progression model consisting of MCF-10A cell lines whose phenotypes ranged from noninvasive to highly invasive. c/EBPalpha expression in breast cancer cells resulted in increased miR-661 expression and reduced MTA1 3'UTR-luciferase activity and MTA1 protein level. We also provide evidence that the introduction of miR-661 inhibited the motility, invasiveness, anchorage-independent growth, and tumorigenicity of invasive breast cancer cells. We believe our findings show for the first time that c/EBPalpha regulates the level of miR-661 and in turn modifies the functions of the miR661-MTA1 pathway in human cancer cells. Based on these findings, we suggest that miR-661 be further investigated for therapeutic use in down-regulating the expression of MTA1 in cancer cells.