Kruppel-like factor 6 (KLF6), a zinc finger transcription factor and tumor suppressor, is induced as an immediate-early gene during hepatic stellate cell (HSC) activation. The paradoxical induction of a tumor suppressor in HSCs during proliferation led us to explore the biology of wildtype KLF6 (KLF6WT) and its antagonistic, alternatively spliced isoform KLF6SV1 in cultured HSCs and animal models. The animal models generated include a global heterozygous KLF6 mouse (Klf6+/-), and transgenic mice expressing either hKLF6WT or hKLF6SV1 under the control of the Collagen α2 (I) promoter to drive HSC-specific gene expression following injury. The rat Klf6 transcript has multiple splice forms that are homologous to those of the human KLF6 gene. Following a transient increase, all rat Klf6 isoforms decreased in response to acute carbon tetrachloride (CCl4) liver injury and culture-induced activation. After acute CCl4, Klf6+/- mice developed significantly increased fibrosis and enhanced fibrogenic messenger RNA (mRNA) and protein expression. In contrast, HSC-specific transgenic mice overexpressing KLF6WT or KLF6SV1 developed significantly diminished fibrosis with reduced expression of fibrogenic genes. Chromatin IP and quantitative reverse-transcription polymerase chain reaction in mouse HSCs overexpressing KLF6WT demonstrated KLF6WT binding to GC boxes in promoters of Colα1 (I), Colα2 (I), and beta-platelet-derived growth factor receptor (β-Pdgfr) with reduced gene expression, consistent with transcriptional repression by KLF6. Stellate cells overexpressing either KLF6WT or KLF6SV1 were more susceptible to apoptotic stress based on poly (ADP-ribose) polymerase (PARP) cleavage. Conclusion: KLF6 reduces fibrogenic activity of HSCs by way of two distinct mechanisms, direct transcriptional repression of target fibrogenic genes and increased apoptosis of activated HSCs. These results suggest that following its initial induction, sustained down-regulation of KLF6 in liver injury may allow de-repression of fibrogenic genes and decreased stellate cell clearance by inhibiting apoptosis.