To investigate the shear behavior of externally prestressed precast concrete segmental beams with both positive and negative moment regions (EPCS P&N beams), eight specimens were subjected to failure under various shear span to depth ratios, moment ratios, and joint types. Throughout the testing process, critical crack initiation and development, joint slippage and opening, failure processes and modes, as well as strains in concrete, stirrups, and external prestressing tendons were meticulously recorded. At the point of failure, the concrete in the shear-compression zone near the top surface of the joint section (closest to the loading point in the positive moment region) or near the bottom surface of the joint section (closest to the interior support in the negative moment region) experienced crushing. Subsequently, the external tendons lost support and underwent elastic retraction, causing the two segments on either side of the critical web-shear crack to slide against each other. The elastic strain energy was released significantly as a consequence of the external tendons retracting, leading to the rupture of stirrups intersecting the failure cracks, followed by flange buckling. Based on the test results, a strut-and-tie model was developed to analyze the shear resistance mechanism, and a detailed flowchart for computing the shear resistance of EPCS P&N beams was established. The predictions from the proposed analytical method were compared with the test results, showing good agreement and confirming the rationality of the proposed shear resistance mechanism.