MicroRNAs are noncoding, endogenous small RNAs that regulate target genes by cleavage of the targeted mRNA or translational repression. We investigated the microRNAome using 2-color microarrays in a highly invasive human breast cancer cell line, MDA-MB-231 (subline 4175) and a noninvasive breast epithelial cell line, MCF10A. We found 13 microRNAs that were up-regulated, and nine were down-regulated significantly in 4175 cells (p < 0.05, -fold change >2) compared with MCF10A cells. Interestingly, miR-27b and its putative target gene, ST14 (suppressor of tumorigenicity 14), had inverse expression pattern in breast cancer cells. The 3'-untranslated region of ST14 contains a regulatory element for miR-27b, and our luciferase experiments indicate that antisense miR-27b enhances ST14 expression in cancer cells. Furthermore, antagomir of miR-27b suppressed cell invasion in 4175 cells, whereas pre-miR-27b stimulated invasion in moderately invasive ZR75 breast cancer cells. In addition, ST14 reduces cell proliferation as well as cell migration and invasion. Analysis of human breast tumors revealed that miR-27b expression increases during cancer progression, paralleling a decrease in ST14 expression. Furthermore, our data indicate that ST14 inhibits cells from entering into S phase by up-regulating p27, which results in down-regulation of cyclin E-CDK2 complexes, suggesting ST14 reduces cell growth through its effects on cell cycle-related proteins. Introduction of miR-27b into ST14-expressing cells did not suppress the effect on cell growth. These findings suggest that ST14 plays an important role in several biological processes, and some effects are not completely dependent on miR-27b regulation.