Catalyzes the first of the four reactions of the mitochondrial fatty acid beta-oxidation (FAO) pathway, 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:17564966, PubMed:21237683, PubMed:24591516, PubMed:32389575). The mitochondrial FAO pathway is the major energy-producing process in tissues and is performed through cycles of four consecutive reactions (PubMed:17564966, PubMed:26474213). Each FAO cycle shortens the fatty acyl-CoA by two carbons, yielding one acetyl-CoA (for the citric acid cycle), one FADH(2), and one NADH (which donate electrons to the respiratory chain for ATP production) (PubMed:17564966, PubMed:26474213). Among the different mitochondrial acyl-CoA dehydrogenases, long-chain specific acyl-CoA dehydrogenase activity overlaps with that of ACADV and ACAD9, acting on saturated and unsaturated acyl-CoAs with 6 to 24 carbons with a preference for 8 to 18 carbons long primary chains (PubMed:17564966, PubMed:21237683, PubMed:32389575, PubMed:8823175). Can use (4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoate as substrate in vitro (which is not primarily used for energy but mainly beta-oxidized in the peroxisomes) (PubMed:17564966, PubMed:26474213). 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). Plays a primary role in FAO in tissues where it is the main long-chain ACAD expressed, such as the lung, specifically in type 2 alveolar cells (responsible for surfactant production) (PubMed:17564966, PubMed:24591516). Probably responsible for beta-oxidation of bulky substrates including branched chain fatty acyl-CoAs and sterol derivatives thanks to its enlarged substrate-binding cavity (PubMed:38839792). {ECO:0000269|PubMed:17564966, ECO:0000269|PubMed:21237683, ECO:0000269|PubMed:24591516, ECO:0000269|PubMed:32389575, ECO:0000269|PubMed:8823175, ECO:0000303|PubMed:26474213, ECO:0000303|PubMed:38839792}. This is the function of ACADL (acyl-CoA dehydrogenase long chain, ENSG00000115361).