To construct a three-dimensional (3D) model of histidine kinase (HK) YycG protein in Streptococcus pneumoniae and to investigate the interaction between YycG and its substrate ADP for the purpose of providing a theoretical basis for YycG selective inhibitor discovery, we constructed a 3D model of YycG protein by homology modeling, and assessed the reliability of the model using ProCheck and Profile_3D software. Besides, the active-site cavity of YycG and the residues key for substrate interaction were analyzed by Autodock4.0. Sequence alignment indicated that the YycG of S. pneumoniae was homologous to that of Thermotoga maritima. The constructed 3D model of YycG adopted a similar folding pattern to the template and the two matched well. The conservative amino acids in the substrate-binding pocket, such as Asn145, Asn149 and Lys152, as well as the hydrophobic residues at the bottom of the pocket played important role in binding and hydrolyzing substrate ADP. We have successfully constructed a reliable model of YycG protein. The model can be used as a starting point for designing antibacterial drugs.