MicroRNAs (miRNA/miR) are a class of small noncoding RNAs implicated in the pathogenesis of various malignancies. In the current study, using micro(RNA) arrays, we found a ubiquitous loss of miR-126 expression in colon cancer lines when compared to normal human colon epithelia. Reconstitution of miR-126 in colon cancer cells resulted in a significant growth reduction as evidenced in clonogenic assays. A search for miR-126 gene targets revealed p85β, a regulatory subunit involved in stabilizing and propagating the phosphatidylinositol 3-kinase (PI3K) signal, as one of the potential substrates. Restoration of miR-126 in cancer cells induced a ≥3-fold reduction in p85β protein levels, with no concomitant change in p85α, a gene that is functionally related to p85β but not a supposed target of miR-126. Additionally, using reporter constructs, we show that the p85β-3′ untranslated region is directly targeted by miR-126. Furthermore, this miR-126 mediated reduction of p85β was accompanied by a substantial reduction in phosphorylated AKT levels in the cancer cells, suggesting an impairment in PI3K signaling. Finally, in a panel of matched normal colon and primary colon tumors, each of the tumors demonstrated miR-126 down-regulation together with an increase in the p85β protein level. Taken together, we propose that miR-126 regulates PI3K signaling partly by targeting p85β, and that the loss of miR-126 may provide a selective growth advantage during colon carcinogenesis. © 2008 Wiley-Liss, Inc.