Islet1 (Isl1) belongs to the LIM homeodomain transcription factor family. Its roles in differentiation of motor neurons and organogenesis of pancreas and heart have been revealed. However, less is known about its regulatory mechanism and the target genes. In this study, we identified interactions between Isl1 and Janus tyrosine kinase (JAK), as well as signal transducer and activator of transcription (Stat)3, but not Stat1 and Stat5, in mammalian cells. We found that Isl1 not only forms a complex with Jak1 and Stat3 but also triggers the tyrosine phosphorylation of Jak1 and its kinase activity, thereby elevating the tyrosine phosphorylation, DNA binding activity, and target gene expression of Stat3. In vivo, the tyrosine-