Human ESCRT-I is a multiprotein complex that plays essential roles in HIV budding and endosomal protein sorting. All ESCRT-I complexes contain three common subunits (TSG101, VPS28, and VPS37), and a fourth subunit of yeast ESCRT-I was recently identified (Mvb12p). We now demonstrate that two related human proteins (MVB12A and MVB12B) constitute the fourth class of metazoan ESCRT-I subunits, despite lacking identifiable sequence homology to Mvb12p. Hydrodynamic studies indicate that soluble human ESCRT-I complexes contain one copy of each of the four subunit types. MVB12 subunits associate with the core region of the binary TSG101-VPS37 complex through conserved C-terminal sequence elements. Both MVB12 depletion and overexpression inhibit HIV-1 infectivity and induce unusual viral assembly defects, including aberrant virion morphologies and altered viral Gag protein processing. Taken together, these studies define the composition of human ESCRT-I complexes and indicate that the MVB12 subunits play a unique role in regulating ESCRT-mediated virus budding.