Honey bees are exposed to various pesticides through pollinating and in-hive Varroa mite control. The most basic method for evaluating pesticide toxicity is the indoor bioassay using worker bees, in which newly emerged adults are matured in incubators for conditioning before use. However, little information is available on the optimum maturation temperature from a toxicological point of view, even though it can affect honey bee responses to pesticides. In this paper, to evaluate the optimal maturation temperature for pesticide toxicity testing, several indices related to the development, gene transcription, and toxicological properties of honey bee adults following maturation at 25, 30, and 35 °C were compared with those of field bees. The body weight and developmental state of hypopharyngeal glands were highest in the bees matured at 30 °C, and the overall transcription profiles of detoxification-related genes in the field bees were closest to those of bees matured at 30 °C, whereas immaturity and features of thermal stress were observed in the 25 °C and 35 °C bee groups, respectively. In the bioassay results, the effects of maturation temperature on the toxic response of honey bees varied significantly depending on the type of pesticide. By considering all the biological and toxicological aspects examined, we confirmed that 30 °C is a recommended maturation temperature for adult honey bee toxicity test.