Gene variants in the alternative pathway of the complement system strongly associate with atypical hemolytic uremic syndrome (aHUS), presumably by predisposing to increased complement activation within the kidney. Complement factor H (CFH) is the major regulator of complement activation through the alternative pathway. Factor H-deficient mice transgenically expressing a mutant CFH protein (Cfh(-/-).FHΔ16-20) that functionally mimics the CFH mutations reported in aHUS patients spontaneously develop thrombotic microangiopathy. To investigate the role of complement C5 activation in this aHUS model, we generated C5-deficient Cfh(-/-).FHΔ16-20 mice. Both C5-sufficient and C5-deficient Cfh(-/-).FHΔ16-20 mice had abnormal C3 deposition within the kidney, but spontaneous aHUS did not develop in any of the C5-deficient mice. Furthermore, although Cfh(-/-).FHΔ16-20 animals demonstrated marked hypersensitivity to experimentally triggered renal injury, animals with concomitant C5 deficiency did not. These data demonstrate a critical role for C5 activation in both spontaneous aHUS and experimentally triggered renal injury in animals with defective complement factor H function. This study provides a rationale to investigate therapeutic inhibition of C5 in human aHUS.