The CCAAT/enhancer binding protein α (C/EBPα) is vital for establishing normal hepatic energy homeostasis and moderating hepatocellular growth. CEBPA loss-of-function mutations identified in acute myeloid leukemia patients support a tumor suppressor role for C/EBPα. Recent work showed reductions of C/EBPα levels in human hepatocellular carcinoma with the reductions correlating to tumor size and progression. We investigated the potential of reactivating c/ebpα expression during hepatic carcinogenesis to prevent tumor cell growth. We have developed a c/ebpα knock-in mouse in which a single-copy c/ebpα is regulated by one allele of the α-fetoprotein (AFP) gene promoter. The knock-in mice are physically indistinguishable from wild-type (WT) controls. However, knock-in animals were found to deposit fetal hepatic glycogen earlier than WT animals. Quantitative real-time PCR confirmed early c/ebpα expression and early glycogen synthase gene activation in knock-in fetuses. We then used diethylnitrosamine to induce hepatocellular carcinoma in our animals. Diethylnitrosamine produced half the number of hepatocellular nodules in knock-in mice as in WT mice. Immunohistochemistry showed reduced C/EBPα content in WT nodules whereas knock-in nodules stained strongly for C/EBPα. The p21 protein was examined because it mediates a C/EBPα growth arrest pathway. Nuclear p21 was absent in WT nodules whereas cytoplasmic p21 was abundant; knock-in nodules were positive for nuclear p21. Interestingly, only C/EBPα-positive nodules were positive for nuclear p21, suggesting that C/EBPα may be required to direct p21 to the cell nucleus to inhibit growth. Our data establish that controlled C/EBPα production can inhibit liver tumor growth in vivo.