Aberrant expression of microRNAs (miRNAs) is frequently associated with a variety of cancers, including breast cancer. We and others have demonstrated that radiation-induced rat mammary cancer exhibits a characteristic gene expression profile and a random increase in aberrant DNA copy number; however, the role of aberrant miRNA expression is unclear. We performed a microarray analysis of frozen samples of eight mammary cancers induced by γ irradiation (2 Gy), eight spontaneous mammary cancers and seven normal mammary samples. We found that a small set of miRNAs was characteristically overexpressed in radiation-induced cancer. Quantitative RT-PCR analysis confirmed that miR-135b, miR-192, miR-194 and miR-211 were significantly up-regulated in radiation-induced mammary cancer compared with spontaneous cancer and normal mammary tissue. The expression of miR-192 and miR-194 also was up-regulated in human breast cancer cell lines compared with noncancer cells. Manipulation of the miR-194 expression level using a synthetic inhibiting RNA produced a small but significant suppression of cell proliferation and upregulation in the expression of several genes that are thought to act as tumor suppressors in MCF-7 and T47D breast cancer cells. Our data suggest that the induction of rat mammary cancer by radiation involves aberrant expression of miRNAs, which may facilitate cell proliferation.