Rabies continues to kill many thousands of people throughout the developing world every year. The murine monoclonal antibody (mAb) 62-71-3 was recently identified for its potential application in rabies post-exposure-prophylaxis (PEP). The purpose here was to establish a plant-based production system for a chimeric mouse-human version of mAb 62-71-3, to characterize the recombinant antibody and investigate at a molecular level its interaction with the rabies virus (RV) glycoprotein. The chimeric mAb 62-71-3 was successfully expressed in Nicotiana benthamiana and functionality was analyzed by antigen binding ELISA and by neutralization of a panel of lyssavirus pseudotypes. The antibody-antigen interaction was investigated using pseudotype virus expressing mutagenized RV glycoproteins. A critical role for antigenic site I of the glycoprotein, in particular for two specific amino acid residues, was identified.