In contrast to UCP1, the primary function of UCP3 is not the dissipation of energy. Rather, several lines of evidence suggest that UCP3 is related to cellular long-chain fatty acid homeostasis. If long-chain fatty acids enter the mitochondrial matrix in their non-esterified form, they cannot be metabolized and may exert deleterious effects. To test the feasibility that UCP3 exports fatty acid anions, we systematically interfered at distinct steps in the fatty acid metabolism pathway, thereby creating conditions in which the entry of (non-esterified) fatty acids into the mitochondrial matrix is enhanced. First, reducing the cellular fatty acid binding capacity, known to increase cytosolic concentrations of non-esterified fatty acids, up-regulated UCP3 5.3-fold. Second, inhibition of mitochondrial entry of esterified long-chain fatty acids up-regulated UCP3 by 1.9-fold. Third, high-fat diets, to increase mitochondrial supply of non-esterified long-chain fatty acids exceeding oxidative capacity, up-regulated UCP3 twofold. However, feeding a similar amount of medium-chain fatty acids, which can be oxidized inside the mitochondrial matrix and therefore do not need to be exported from the matrix, did not affect UCP3 protein levels. These data are compatible with a physiological function of UCP3 in facilitating outward transport of long-chain fatty acid anions, which cannot be oxidized, from the mitochondrial matrix.