Mitochondrial β-oxidation is crucial for maintaining cellular health when lipid supply to the cell is high. Yet, there is evidence that links organ pathology to mitochondrial fatty acid oxidation, per se. The associated pathologies range from insulin resistance and poor recovery from ischemia-reperfusion episodes in various organs, to the development of renal fibrosis. Often, studies into the mechanistic bases of such pathologies operate under a paradigm of “insufficient” or “excessive” FAO as a causative factor. However, “classic” studies, the literature on genetic β-oxidation disorders and the recent use of sophisticated proteomics platforms to detect post-translational modifications all suggest that the abundance of fatty acyl-CoA can be regulated within the mitochondrial matrix. In terms of mechanisms, pyruvate dehydrogenase kinases, enabling entry into the Krebs cycle of β-oxidation-derived acetyl-CoA, were identified in the earlier literature. Several years then passed before interest was re-invigorated with genetic mouse studies on the mitochondrial sirtuins (protein deacylases) and on carnitine-o-acyltransferase (CrAT; converts short-chain acyl-CoAs into carnitine esters). Thus how β-oxidation, particularly acyl- and acetyl-CoA abundance, is regulated is again emerging as an important mechanism to mitigate potentially deleterious effects of high lipid supply. Yet, the full complement of matrix mechanisms that undertake this regulation, and whether and how they interact, are major questions. We have identified a new mechanism that regulates acyl-CoA abundance in the mitochondrial matrix: Acot2. The existence in the matrix of acyl-CoA thioesterases (Acot) that hydrolyze acyl-CoA esters into an acyl chain and free CoA has been known for many years, and these Acots have been cloned. However, their biological role, especially in highly aerobic tissues such as the skeletal muscle, has remained unexplored. Moreover, given the existence of CrAT, a role for Acots in β-oxidation regulation might seem redundant. Yet our initial studies using a new mouse model of the matrix enzyme, Acot2, clearly suggest the hypothesis that Acot2 regulates entry of long-chain fatty acyl-CoA into β-oxidation, or siphons it off from β-oxidation. The major substrates for Acot2 are C14:0-, C16:0- and C16:1-CoA. In this way, Acot2 could function as a control point for β-oxidation, a concept that would expand how we think about β-oxidation regulation. The associated phenotypes in the Acot2 depleted mice consuming a standard chow diet generally point to a beneficial metabolic role for Acot2 in muscle and liver. This suggests that a matrix-resident mechanism that limits long-chain fatty acyl-CoA abundance and/or ensures CoA availability in the mitochondrial matrix is protective.