As a melanosome-associated transmembrane glycoprotein, GPNMB plays an important role in numerous cell types, as well as in tumors. Producing a high specificity and affinity monoclonal antibody against human GPNMB provides an important tool to study the function of GPNMB protein. In this study, monoclonal antibodies to GPNMB were obtained by immunizing BALB/c mice with purified GST-GPNMB emulsified in Freund's adjuvant. Three monoclonal antibodies with high specificity and affinity were obtained. The titers of anti-serum were 1:10,000, 1:8000, and 1:3000, respectively. Western blot and immunohistochemistry experiments were used to characterize the antibody. The anti-GPNMB antibodies G203 and F105 had high affinities (G203 around 2.7 × 10(-8) M and F105 around 1.6 × 10(-8) M, respectively) for the GPNMB antigen. However, M306 had a low binding activity to GPNMB. The results of Western blot and immunohistochemistry experiments showed that the antibodies could bind human GPNMB antigen. The monoclonal antibodies provided good tools for further studying functional characterization of GPNMB.