Human sine oculis homeobox homolog (SIX) 1 contains a homeodomain (HD), which is important for binding to DNA. In this study, we carried out structural studies on the HD of human SIX1 using nuclear magnetic resonance (NMR) spectroscopy. Its secondary structures and dynamics in solution were explored. HD is well-structured in solution, and our study shows that it contains three α-helices. Dynamics study indicates that the N- and C-terminal residues of HD are flexible in solution. HD of human SIX1 exhibits molecular interactions with a short double-strand DNA sequence evidenced by the 1H-15N-heteronuclear single quantum correlation (HSQC) and 19F-NMR experiments. Our current study provides structural information for HD of human SIX1. Further studies indicate that this construct can be utilized to study SIX1 and DNA interactions.