Ivermectin and moxidectin are the most widely administrated anthelmintic macrocyclic lactones (MLs) to treat human and animal nematode infections. Their widespread and frequent use has led to high level of resistance to these drugs. Although they have the same mode of action, differences in terms of selection for drug resistance have been reported. Our objective was to study and compare changes occurring upon ivermectin or moxidectin selection in the model nematode Caenorhabditis elegans C. elegans worms were submitted to stepwise exposure to increasing doses of moxidectin. Sensitivity to MLs of moxidectin-selected worms was determined in a larval development assay and compared with wild-type and ivermectin-selected strains. Selection with either ivermectin or moxidectin led to acquired tolerance to ivermectin, moxidectin and eprinomectin. Importantly, moxidectin was the most potent ML in both ivermectin- and moxidectin-selected strains. Interestingly, this order of potency was also observed in an Haemonchus contortus resistant isolate. In addition, ivermectin- and moxidectin-selected strains displayed a constitutive overexpression of several genes involved in xenobiotic metabolism and transport. Moreover, verapamil potentiated sensitivity to ivermectin and moxidectin, demonstrating that ABC transporters play a role in ML sensitivity in ML-selected C. elegans strains. Finally, both ivermectin- and moxidectin-selected strains displayed a dye filling defective phenotype. Overall, this work demonstrated that selection with ivermectin or moxidectin led to cross-resistance to several MLs in nematodes and that induction of detoxification systems and defects in the integrity of amphidial neurons are two mechanisms that appear to affect the responsiveness of worms to both ivermectin and moxidectin.