Mixl1 is a paired-type homeodomain protein that plays a crucial role in morphogenesis and endoderm differentiation in the murine embryo. To understand how Mixl1 directs embryogenesis, we studied the regulation of Mixl1 expression at a transcriptional level. In HepG2 cells, a genomic fragment encompassing the Mixl1 promoter conferred strong TGF-beta-induced transcription that was dependent on the presence of the DNA-binding protein FoxH1. Further analysis of the Mixl1 promoter identified a proximal response element (PRE) containing SMAD- and FoxH1-binding sites required for TGF-beta responsiveness. The PRE was also responsive to signalling by Nodal, a TGF-beta ligand required for normal embryonic patterning. These results demonstrate for the first time a functional role for TGF-beta ligands in regulation of mammalian Mixl1, identify FoxH1 as an essential transcriptional co-activator, and implicate Nodal as the embryonic regulator of Mixl1 in mesendoderm morphogenesis.