The complement system is regulated by inhibitors such as factor I (FI), a serine protease that degrades activated complement factors C4b and C3b in the presence of specific cofactors. Mutations and polymorphisms in FI and its cofactors are associated with atypical hemolytic uremic syndrome (aHUS). All 14 complement factor I mutations associated with aHUS analyzed in this study were heterozygous and generated premature stop codons (six) or amino acid substitutions (eight). Almost all of the mutants were expressed by human embryonic kidney 293 cells but only six mutants were secreted into the medium, three of which were at lower levels than WT. The remaining eight mutants were not secreted but sensitive to deglycosylation with endoglycosidase H, indicating that they were retained early in the secretory pathway. Six secreted mutants were purified and five of them were functionally altered in degradation of C4b/C3b in the fluid-phase in the presence of various cofactors and on endothelial cells. Three mutants cleaved surface-bound C3b less efficiently than WT. The D501N mutant was severely impaired both in solution and on surface irrespective of the cofactor used. In conclusion, mutations in complement factor I affect both secretion and function of FI, which leads to impaired regulation of the complement system in aHUS.