MicroRNAs are master gene regulators that can also be under the control of transcriptional regulation. We have previously shown that miR-145 is a tumor suppressor capable of silencing c-Myc and the tumor suppressor p53 induces miR-145 by directly binding to the miR-145 promoter, demonstrating the role of miR-145 in p53-mediated c-Myc repression. However, little is known as to why miR-145 is often downregulated in tumors. In this study, we identify CCAAT/enhancer binding protein beta (C/EBP-β) as a negative regulator for miR-145 expression by direct interaction with the putative C/EBP-β binding site in the miR-145 promoter. In the wild-type p53 background, C/EBP-β counteracts the ability of p53 to induce miR-145. Moreover, C/EBP-β is able to suppress miR-145 in the mutant p53 background, suggesting the p53 independent regulation of miR-145. Of interest, both the large isoform (LAP-2) and the small isoform (LIP) of C/EBP-β can exert suppressive function for miR-145. Finally, we further show that, like serum starvation and PI3K inhibitor LY29, the antioxidant resveratrol suppresses pAkt and phosphorylation of C/EBP-β and at the same time, it induces miR-145. Together, these results suggest a miR-145 regulatory system involving the Akt and C/EBP-β, which may contribute to the downregulation of miR-145 in cancer cells.