Respiratory complex I (NADH:ubiquinone oxidoreductase) is a large mitochondrial inner membrane enzyme consisting of 45 subunits and 8 iron-sulfur (Fe/S) clusters. While complex I dysfunction is the most common reason for mitochondrial diseases, the assembly of complex I and its Fe/S cofactors remains elusive. Here, we identify the human mitochondrial P-loop NTPase, designated huInd1, that is critically required for the assembly of complex I. huInd1 can bind an Fe/S cluster via a conserved CXXC motif in a labile fashion. Knockdown of huInd1 in HeLa cells by RNA interference technology led to strong decreases in complex I protein and activity levels, remodeling of respiratory supercomplexes, and alteration of mitochondrial morphology. In addition, huInd1 depletion resulted in massive decreases in several subunits (NDUFS1, NDUFV1, NDUFS3, and NDUFA13) of the peripheral arm of complex I, with the concomitant appearance of a 450-kDa subcomplex representing part of the membrane arm. By a novel radiolabeling technique, the amount of iron associated with complex I was also shown to reflect the dependence of this enzyme on huInd1 for assembly. Together, these data identify huInd1 as a new assembly factor for human respiratory complex I with a possible role in the delivery of one or more Fe/S clusters to complex I subunits.