Imidacloprid (IMD) is a neonicotinoid insecticide widely used in various crops and animals for pest control. IMD is rapidly absorbed by the gastrointestinal tract, being rapidly and evenly distributed in the organs and tissues. The highest concentrations were observed in the elimination organs: liver and kidneys. The liver is the main organ involved in the biotransformation of exogenous substances (xenobiotics), with the capacity to convert hydrophobic compounds into water soluble metabolites, which are more easily eliminated by the organism. Studies have been conducted on the toxic effects of IMD on animals, causing damage to the liver. In this sense, the objective of this study was to evaluate the mechanisms involved in the toxicity of IMD on the bioenergetics of mitochondria and isolated hepatocytes of rats and its actions on the metabolism of carbohydrates and proteins in liver of rats in perfusion. In isolated mitochondria, IMD promoted a dose-dependent decrease in the state 3 of mitochondrial respiration and ATP levels, without affecting mitochondrial membrane potential. Subsequent experiments measuring oxygen consumption have shown that IMD does not affect the electron transport chain and that its effect is similar to that of oligomycin (FoF1-ATP synthase inhibitor) and/or atracytoside (ANT adenine nucleotide translocator inhibitor). In the perfusion rat liver IMD inhibited the activity of FoF1-ATP synthase in freeze/thaw-disrupted mitochondria and partially inhibited the depolarization of the membrane potential induced by ADP. These results indicate that IMD affects in mitochondrial bioenergetics by inhibiting FoF1-ATP synthase. In experiments with isolated hepatocytes respiration results were similar to those found in isolated mitochondria, but IMD affected the intracellular production of ATP and induced cell death only in hepatocytes isolated from rats previously treated with dexamethasone, a cytochrome P450 activator. IMD also inhibited the production of glucose by gluconeogenesis. These results suggest that IMD toxicity may be associated with changes in cellular energy metabolism with the enzyme FoF1-ATP synthase being the main target of the toxic action of this insecticide, and that the metabolites formed in the biotransformation of the IMD may be more toxic than the IMD itself.