Mnt is a Max-interacting transcriptional repressor that has been hypothesized to function as a Myc antagonist. To investigate Mnt function we deleted the Mnt gene in mice. Since mice lacking Mnt were born severely runted and typically died within several days of birth, mouse embryo fibroblasts (MEFs) derived from these mice and conditional Mnt knockout mice were used in this study. In the absence of Mnt, MEFs prematurely entered the S phase of the cell cycle and proliferated more rapidly than Mnt(+/+) MEFs. Defective cell cycle control in the absence of Mnt is linked to upregulation of Cdk4 and cyclin E and the Cdk4 gene appears to be a direct target of Mnt-Myc antagonism. Like MEFs that overexpress Myc, Mnt(-/-) MEFs were prone to apoptosis, efficiently escaped senescence and could be transformed with oncogenic Ras alone. Consistent with Mnt functioning as a tumor suppressor, conditional inactivation of Mnt in breast epithelium led to adenocarinomas. These results demonstrate a unique negative regulatory role for Mnt in governing key Myc functions associated with cell proliferation and tumorigenesis.