Kruppel-like factor (KLF) 6 is a tumor-suppressor gene functionally inactivated by loss of heterozygosity, somatic mutation and/or alternative splicing that generates a dominant-negative splice form, KLF6-SV1. Wild-type KLF6 (wtKLF6) expression is decreased in many human malignancies, which correlates with reduced patient survival. Additionally, loss of the KLF6 locus in the absence of somatic mutation in the remaining allele occurs in a number of human cancers, raising the possibility that haploinsufficiency of the KLF6 gene alone contributes to cellular growth dysregulation and tumorigenesis. Our earlier studies identified the cyclin-dependent kinase inhibitor p21 as a transcriptional target of the KLF6 gene in cultured cells, but not in vivo. To address this issue, we have generated two genetic mouse models to define the in vivo role of KLF6 in regulating cell proliferation and p21 expression. Transgenic overexpression of KLF6 in the liver resulted in a runted phenotype with decreased body and liver size, with evidence of decreased hepatocyte proliferation, increased p21 and reduced proliferating cell nuclear antigen expression. In contrast, mice with targeted deletion of one KLF6 allele (KLF6+/-) display increased liver mass with reduced p21 expression, compared to wild type littermates. Moreover, in primary hepatocellular carcinoma samples, there is a significant correlation between wtKLF6 and p21 mRNA expression. Combined, these data suggest that haploinsufficiency of the KLF6 gene may regulate cellular proliferation in vivo through decreased transcriptional activation of the cyclin-dependent kinase inhibitor p21.