To study the seismic behavior of prefabricated reinforced concrete column-steel beam (RCS) hybrid structures, a cyclic loading test on a novel RCS joint was conducted to investigate the failure modes, ductility, and energy dissipation. The moment-rotation relationship model of the joint was proposed according to the test. Furthermore, the model of a 3-bay, 5-story semi-rigid prefabricated RCS frame was established in SAP2000 software. The modal analysis and dynamic elastic–plastic analysis were carried out on the semi-rigid and rigid RCS frames respectively to study the effect of semi-rigid connections on the seismic performance of RCS structures. Analysis results show that the failure mode of the prefabricated RCS joint meets the strong column-weak beam requirement. The ‘bow’-shaped hysteretic curve indicates the joint has good seismic performance with good ductility and energy dissipation capacity. Compared to rigid RCS structures, semi-rigid connections lead to a reduction in the overall stiffness and an increase in the natural vibration periods of the structure. Under the action of the earthquake, the base shear force of the semi-rigid RCS structure is smaller than that of the rigid RCS structure. The maximum inter-story drift angles and top displacement of semi-rigid RCS structures will increase, but still meet the requirements of the seismic standard.