How proliferative and inhibitory signals integrate to control liver regeneration remains poorly understood. A screen for antiproliferative factors repressed after liver injury identified transducer of ErbB2.1 (Tob1), a member of the PC3/BTG1 family of mito-inhibitory molecules as a target for further evaluation. Tob1 protein decreases after 2/3 hepatectomy in mice secondary to posttranscriptional mechanisms. Deletion of Tob1 increases hepatocyte proliferation and accelerates restoration of liver mass after hepatectomy. Down-regulation of Tob1 is required for normal liver regeneration, and Tob1 controls hepatocyte proliferation in a dose-dependent fashion. Tob1 associates directly with both Caf1 and cyclin-dependent kinase (Cdk) 1 and modulates Cdk1 kinase activity. In addition, Tob1 has significant effects on the transcription of critical cell cycle components, including E2F target genes and genes involved in p53 signaling. We provide direct evidence that levels of an inhibitory factor control the rate of liver regeneration, and we identify Tob1 as a crucial check point molecule that modulates the expression and activity of cell cycle proteins.