Objectives To characterize a pyrazinamidase from non-pathogen Pseudonocardia carboxydivorans. Results A pyrazinamidase gene pncA encoding a 23-kDa protein PncA-Pse from P. carboxydivorans was over-expressed in Escherichia coli and characterized. This PncA-Pse can convert both pyrazinamide and nicotinamide efficiently with the optimal pH and temperature of pH 8.5 and 45 degrees C, respectively. Although ferrous iron and manganese were detected in PncA-Pse, the enzymatic activity is not affected by EDTA with the final concentration of 10 mM. Moreover, the enzymatic activity was not significantly affected with the addition of several metal ions, respectively. Based on the structure modeling, the 61st histidine which is associated with the metal binding, was mutated into alanine to get mutant H61A. No activity, iron and manganese were detected for H61A, which implies that PncA-Pse is a metal enzyme with resistance of the metal ion chelator EDTA, which is different from the previous reports. Conclusion This is the first characterized pyrazinamidase from the genus Pseudonocardia, a non-pathogen.