The endoribonuclease RNase-L is the terminal component of an interferon (IFN) -regulated RNA decay pathway known as the 2'-5'-oligoadenylate (2-5A) system whose established functions include antimicrobial and tumor suppressive activities. RNase-L enzymatic activity requires the binding of the small molecule 2-5A, leading to RNase-L dimerization and cleavage of single-stranded RNA. RNase-L levels are controlled post-transcriptionally by the 3'-untranslated region (3'UTR) of its messenger RNA (mRNA), which exerts a strong negative regulatory effect on RNase-L expression. MicroRNAs (miRNAs) are a class of small noncoding RNAs that repress expression of target mRNAs by binding to regions of complementarity often located in their 3'UTRs. The miR-29 family has been shown to play a tumor suppressive role in several cancers, including chronic myelogenous leukemia (CML), and has many known oncogenic targets. Here, we report that the miR-29 family represses expression of RNase-L protein across several cell types. Using a luciferase reporter, we showed that miR-29 acts via four target sites within the RNASEL 3'UTR. Mutation of all sites is required for abrogation of miR-29 repression. MiR-29 repression of RNase-L expression modulates its biologic activities, such as IFN-induced protection against encephalomyocarditis virus infection. In light of the reported tumor suppressive role of miR-29 in K562 CML cells and miR-29 repression of RNase-L expression in these cells, we generated K562 cells with stable Rnase-L knockdown and demonstrated that loss of RNase-L expression inhibits cell proliferation in vitro as well as tumor growth in a nude mouse xenograft model. Our findings identify a previously unknown miRNA regulator of RNase-L expression and support a novel oncogenic role for RNase-L in CML and potentially other malignancies.