Characteristic impedance and its definition are revisited and discussed for microstrip line with a self-calibrated threedimenional (3-D) method of moments (MoM). This 3-D MoM accommodates a scheme called short-open calibration (SOC) so that potential parasitic effects brought by the impressed voltage excitation and other relevant factors can be effectively removed. In this way, the characteristic impedance can be accurately defined through a relationship between equivalent voltage and current on the two sides of a microstrip line having a finite length. Simulated results are compared with the Jansen’s twodimensional (2-D) and Rautio’s 3-D definition.