In humans, the Hox genes are distributed into four linkage groups, HoxA, HoxB, HoxC, and HoxD, which locate on chromosomes 7, 17, 12, and 2, respectively. HoxA1 is thought to be involved in the placement of hindbrain segments in the proper location along the anterior-posterior axis during development. Homeodomain (HD) protein has 60 amino acids which are highly conserved in α1 and α3 helices. The N-terminal arm (L1) and loop regions (L2 and L3) between the helices are also well conserved. Interestingly, the second and third residues in L1 loop are conserved as the positive charged residues, Lys(K) or Arg(R), among almost all Hox proteins. However, the HoxA1 has residues Asn(N) and Ala(A) in position 2 and 3 instead of K and R. In order to understand molecular mechanism of DNA recognition of HoxA1, we have performed NMR experiments on the homeodomain of HoxA1 (HoxA1-HD) complexed with 10-bp consensus DNA and 10-bp single variant DNA duplexes, at a variety of DNA-protein molar ratios. In addition, to clarify the role of residues 2 and 3 in Loop 1, we prepared HoxA1-KR mutant in which residues N2 and A3 are replaced by K2 and R3, respectively, and compared its structural feature in a complex with DNA with those of wild-type HoxA1-HD. We also determined the thermodynamic parameters for each DNA binding of WT HoxA1-HD and HoxA1-KR mutant using Isothermal titration calorimetry (ITC). Our study provides an insight into the role of residues N2 and A3 during target DNA recognition of HoxA1.