Calibration of an X-ray imaging system involves estimation of the 3D geometrical configuration of the system components. Here, we propose a calibration method for stereo X-ray imaging systems, which capture two X-ray images at two orthogonal positions. The calibration parameters include the relation between image coordinates and world coordinates, the relative 3D positions of the X-ray source and detector, and the rotational axis of the stereo X-ray system. The average error of the proposed calibration method was determined to be approximately 0.03% in evaluation tests.