Dinoseb is a highly toxic pesticide of the dinitrophenol group. Its use has been restricted, but it can still be found in soils and waters in addition to being a component of related pesticides that, after ingestion by humans or animals, can originate the compound by enzymatic hydrolysis. As most dinitrophenols, dinoseb uncouples oxidative phosphorylation. In this study, distribution, lipid bilayer affinity and kinetics of the metabolic effects of dinoseb were investigated, using mainly the isolated perfused rat liver, but also isolated mitochondria and molecular dynamics simulations. Dinoseb presented high affinity for the hydrophobic region of the lipid bilayers, with a partition coefficient of 3.75 × 104 between the hydrophobic and hydrophilic phases. Due to this high affinity for the cellular membranes dinoseb underwent flow-limited distribution in the liver. Transformation was slow but uptake into the liver space was very pronounced. For an extracellular concentration of 10 μM, the equilibrium intracellular concentration was equal to 438.7 μM. In general dinoseb stimulated catabolism and inhibited anabolism. Half-maximal stimulation of oxygen uptake in the whole liver occurred at concentrations (2.8–5.8 μM) at least ten times above those in isolated mitochondria (0.28 μM). Gluconeogenesis and ureagenesis were half-maximally inhibited at concentrations between 3.04 and 5.97 μM. The ATP levels were diminished, but differently in livers from fed and fasted rats. Dinoseb disrupts metabolism in a complex way at concentrations well above its uncoupling action in isolated mitochondria, but still at concentrations that are low enough to be dangerous to animals and humans even at sub-lethal doses.