The honey bee, Apis mellifera, provides about $15 billion USD in crop value each year in the U.S. alone in the form of pollination services. Since 2006, commercial beekeepers have reported an average annual overwintering loss of about 28.6% of all managed colonies. There are many factors that are thought to contribute to colony loss including bee-specific pests (e.g. the Varroa destructor mite), bee-specific pathogens (e.g. Nosema fungus), modern beekeeping practices, diminished genetic variability, poor queens, climate change, and exposure to agricultural pesticides. While not the single cause of colony loss, the neonicotinoid insecticides elicit sublethal effects to honey bees that could increase their sensitivities to other stressors that affect colony health. Previous studies found that honey bees have differential sensitivities to the neonicotinoid insecticide imidacloprid, which suggest a mechanism of tolerance to the insecticide in certain colonies. In this study, I examined the imidacloprid sensitivity of honey bees collected from different colonies. After determining a range of LC50 values in the tested colonies, I examined the metabolic detoxification activities of honey bees collected from two colonies that represented the highest and lowest LC50 values, between which there was a 36-fold difference in their LC50 values. I discovered that of the three main families of metabolic detoxification enzymes, general esterases, cytochrome P450 monooxygenases, and glutathione S-transferases (GSTs), a reduction of GST activity with diethyl maleate (DEM) significantly increased imidacloprid-mediated mortality to the honey bees. A comparative analysis of GST kinetic activity from imidacloprid-susceptible and -insensitive honey bees revealed a lower bimolecular inhibition rate constant (ki) for imidacloprid-insensitive individuals (5.07 ± 0.098 nmol/min/mg protein) compared to the imidacloprid-sensitive honey bees (17.23 ± 1.235 nmol/min/mg protein). The IC50 of DEM estimated for bees from each colony showed that the imidacloprid-susceptible honey bees possess a higher IC50 (10 μM) than that of the tolerant honey bees (3 μM). These data suggest that the GSTs in the imidacloprid-tolerant honey bees might be a more efficient detoxification mechanism for the conjugation and elimination of imidacloprid, or imidacloprid metabolites, compared to that of imidacloprid-susceptible honey bees. Therefore, I hypothesize that the differences in metabolic detoxification enzyme activities of honey bees collected from different colonies can result in the differential toxicities of honey bees exposed to neonicotinoid insecticides, such as imidacloprid. However, a thorough examination of imidacloprid detoxification in honey bees is warranted to confirm this hypothesis.