Angiotensin II (Ang II) plays a major role in the pathogenesis of cardiac fibrosis in hypertension. It is known that Ang II induces TGF-beta 1 expression. How transcription mediates Ang II-induced TGF-beta 1 expression, as well as its contribution to cardiac fibrosis, is unknown. We studied the role of Kruppel-like family transcription factors in Ang II-induced myofibroblast formation. We found that among the Kruppel-like family members, Kruppel-like factor 4 (Klf4) was the highest expressed form in isolated cardiac fibroblasts after Ang II treatment. Klf4 increased expression of alpha-SMA and collagen, as well as increased myofibroblast formation. ChIP assays showed that Klf4 specifically bound to the TGF-b1 promoter. Deletion and mutagenesis analysis showed that the sites at -184 similar to-180 bp and -45 similar to-41 bp in the TGF-beta 1 promoter were responsible for Klf4 transactivation of the TGF-beta 1 promoter. Our studies demonstrate that Klf4 plays a pivotal role in Ang II-induced cardiac myofibroblast differentiation and collagen synthesis through transcriptional upregulation of TGF-beta 1.