Abstract: A theory and algorithms of camera calibration based on two parallel line segments are proposed. 1) If the length ratio of the two parallel segments is known, then from its projections across n images, a) 2(n−1) quadratic constraint equations on camera internal parameters can be obtained; b) (5n−6) constraint equations can be obtained if additionally the epipoles across n images are known, among which 3(n − 1) are quadratic, (n − 2) third degree, and (n − 1) fourth degree. 2) If the length ratio of the two parallel line segments is unknown, (2n − 3) constraint equations are derived from n images, among which (n − 2) are of fourth degree, and (n − 1) of sixth degree. Based on the above theoretical results, two practical algorithms of camera calibration are also proposed. Experimental results on synthetic and real images validate the proposed theory and algorithms. The results in this paper seem to be not only of theoretical significance, but also of wide applicability as parallel line segments are not rare in any man-made scene.