Mitochondrial complex I harbors 7 mitochondrial and 38 nuclear-encoded subunits. Its biogenesis requires the assembly and integration of distinct intermediate modules, mediated by numerous assembly factors. The mitochondrial complex I intermediate assembly (MCIA) complex, containing assembly factors NDUFAF1, ECSIT, ACAD9, and TMEM126B, is required for building the intermediate ND2-module. The role of the MCIA complex and the involvement of other proteins in the biogenesis of this module is unclear. Cell knockout studies reveal that while each MCIA component is critical for complex I assembly, a hierarchy of stability exists centered on ACAD9. We also identify TMEM186 and COA1 as bona fide components of the MCIA complex with loss of either resulting in MCIA complex defects and reduced complex I assembly. TMEM186 enriches with newly translated ND3, and COA1 enriches with ND2. Our findings provide new functional insights into the essential nature of the MCIA complex in complex I assembly. Display omitted •The MCIA complex is required for stability of mtDNA-encoded ND2•Assembly factors show a hierarchy of stability centered on ACAD9•TMEM186 interacts strongly with newly synthesized ND3•COA1 interacts with newly translated ND2 and is dispensable for complex IV assembly Formosa et al. investigate the function of the MCIA complex in complex I assembly. They demonstrate the requirement of individual components for the formation of complex I intermediates and assembly of the final enzyme. Finally, they characterize the involvement of TMEM186 and COA1 in this process.