The cattle tick Rhipicephalus microplus is one of the most important ectoparasites with great sanitary and economic impact for cattle rearing worldwide. Ivermectin is commonly used to control tick populations, but its use over the last 30 years has led to the development of resistant populations of R. microplus, and a concomitant loss of efficacy. In this context, we aimed to determine the metabolic mechanisms that contribute to ivermectin resistance in a resistant strain of this species. We performed lethal time bioassays with inhibitors of detoxifying enzymes and xenobiotic transporters (four detoxification pathways) using two strains of ticks: a susceptible strain, Mozo, and a resistant strain, Juarez. We used four inhibitors to test the involvement of different families of proteins responsible for detoxification of ivermectin, namely cytochrome P450, esterases, glutathione-S-transferase, and ATP Binding Cassette Transporters. We calculated the synergistic factor for each inhibitor and strain. To different degrees, all tested inhibitors altered the mortality rates in the strain Juarez, indicating that multiple mechanisms are responsible for the resistant phenotype. Detoxification mechanisms mediated by ABC transporters were observed to be the most important. Esterases, glutathione-S-transferases, and cytochrome-oxidases played less important roles in detoxification.