Decay-accelerating factor (DAF) and complement receptor 1-related gene/protein y (Crry) are two membrane-anchored complement regulatory proteins in rodent. Although both proteins are broadly distributed and exert complement regulation at the same steps of the complement cascade, DAF knockout mice are viable whereas Crry knockout mice die in utero as a result of maternal complement attack. The latter outcome has prevented the dissection of overlapping functions of DAF and Crry in adult mouse tissues in vivo. By crossing female DAF(-/-)/Crry(-/-)/C3(-/-) mice with male DAF(-/-)/Crry(+/-)/C3(+/-) mice, we circumvented maternal complement attack during fetal development and generated viable DAF(-/-)/Crry(-/-)/C3(+/-) mice to address the consequence of DAF/Crry double deficiency. DAF(-/-)/Crry(-/-)/C3(+/-) mice were born at the expected frequency and survived to adulthood. However, they were found to have greatly reduced systemic complement activity due, at least in part, to spontaneous C3 activation and consumption. Plasma C3 proteins in DAF(-/-)/Crry(-/-)/C3(+/-) mice were 30% of that of wild-type mice, and serum complement activity, as assessed by zymosan and immune complex C3 opsonization assays, was 90% reduced in DAF(-/-)/Crry(-/-)/C3(+/-) mice. Remarkably, despite greatly reduced systemic complement activity, DAF(-/-)/Crry(-/-)/C3(+/-) mice developed more severe proteinuria after induction of nephrotoxic serum nephritis as compared with DAF(-/-)/Crry(+/-)/C3(+/-) and DAF(-/-)/Crry(-/-)/C3(-/-) littermate controls. The results highlight the critical and overlapping role of Crry and DAF in vivo in preventing complement activation and tissue injury.