The function of ACAD9 (acyl-CoA dehydrogenase family member 9, Ensembl gene identifier ENSG00000177646) is as follows. Together with NDUFAF1 and ECSIT, forms part of the mitochondrial complex I (MCIA),which is required for the biogenesis of respiratory Complex I (CI) and is therefore crucial for the activation of the oxidative phosphorylation system (PubMed:20816094, PubMed:24158852, PubMed:32320651, PubMed:38086790). ECSIT binding triggers a large conformational change, switching ACAD9 from a fatty acid oxidation (FAO) enzyme to a CI assembly factor (PubMed:38086790). The function in CI assembly is independent of the fatty acid oxidation (FAO) activity of the protein (PubMed:24158852). As FAO enzyme, it catalyzes the first step in mitochondrial FAO, which consists in the proR-proR stereospecific alpha, beta-dehydrogenation of fatty acyl-CoA thioesters using the electron transfer flavoprotein (ETF) as their physiologic electron acceptor, resulting in the formation of trans-2- enoyl-CoA ((2E)-enoyl-CoA) (PubMed:12359260, PubMed:16020546, PubMed:17564966, PubMed:21237683, PubMed:24158852). Its preferred substrates are both saturated and unsaturated long-chain acyl-CoA substrates, with optimum activity toward the latter (PubMed:12359260, PubMed:16020546, PubMed:17564966, PubMed:21237683, PubMed:24158852). In addition, based on its established catalytic mechanism, and combined genetic interaction or mutant phenotype evidence, it is predicted to act also on substrates that have not been tested experimentally but are metabolized by mitochondrial FAO, including long-chain unsaturated fatty acids such as linoleate (9Z,12Z-octadecadienoate), linolenate (9Z,12Z,15Z-octadecatrienoate), and others (PubMed:26474213). Among the different mitochondrial acyl-CoA dehydrogenases, its FAO activity overlaps with that of ACADV and ACADL, but plays a primary role in tissues where it is the main long-chain ACAD expressed, such as the central nervous system (PubMed:16020546, PubMed:17564966, PubMed:24158852, PubMed:25721401, PubMed:26474213). It differs significantly from ACADVL in the use of polyunsaturated substrates in vitro, especially docosahexaenoic acid (which is not primarily used for energy but mainly beta-oxidized in the peroxisomes) (PubMed:16020546, PubMed:17564966). {ECO:0000269|PubMed:12359260, ECO:0000269|PubMed:16020546, ECO:0000269|PubMed:17564966, ECO:0000269|PubMed:20816094, ECO:0000269|PubMed:21237683, ECO:0000269|PubMed:24158852, ECO:0000269|PubMed:25721401, ECO:0000269|PubMed:32320651, ECO:0000269|PubMed:38086790, ECO:0000303|PubMed:26474213}.