In this paper, a novel prefabricated reinforced concrete (PC) self-sustaining beam-column connection for moment-resisting frames was developed to achieve the targets of short erection time, high construction efficiency, low-cost and satisfactory seismic performance. The connection design eliminates the need of temporary supports for the PC beams and slabs during the assembly process in site, and reduces the amount of lateral supports for PC multi-storey columns and formwork for cast-in-place concrete. As the designed thickness of PC U-shells at the beam ends was about 1/3 of the beam width, there could be a marked effect on the achieved integrity of such connections, especially under seismic loading. To investigate the seismic performance of this PC connection, five large-scale PC self-sustaining beam-column connections specimens and one reference conventional RC connection were designed and tested under reverse cyclic loading. The test parameters included the length and area of the flexural reinforcing bars placed at the bottom of PC U-shells, and the anchorage measures (stirrups) inside the PC U-shell. The five precast specimens exhibited similar crack distributions and failure patterns due to the gap-opening between the PC beams and column surface, which was attributed to the reduced effective width and depth of beam cross-section. The test results showed that the use of longer flexural reinforcing bars had little influence on the load-carrying capacity, but contributed to the initial stiffness and energy dissipation capacity. The load-carrying capacity increased by 24% when the area of flexural reinforcing bars increased by 50% in the U-shell region. The incorporation of stirrups in the overlapping region of beam flexural reinforcing bars and longitudinal rebars improved their bond-slip behaviour in specimen PC-S. Compared with specimen PC-C, the energy dissipation capacity of specimen PC-S was improved by 16.5%. Finally, the failure pattern and load-carrying capacity of the PC specimens were analysed and discussed using a simplified mechanical model.