As one of the most important eukaryotic DNA-binding motifs, the homeodomain has been identified in over one thousand proteins. Transcription factors containing homeodomains play critical roles in diverse biological processes and are often implicated in human genetic disorders. The human Pitx2 protein is a central downstream transcription factor in both the TGF-β and Wnt/β-catenin signaling pathways, and involved in the regulation of cell differentiation, cell pattern formation and tissue maintenance. To better understand how Pitx2 regulates its target genes, we are investigating the mechanisms that confer DNA-binding specificity on its homeodomain. Here, we report complete (1)H, (13)C and (15)N protein chemical shift assignments of the human Pitx2 homeodomain in complex with a non-consensus DNA sequence.