Insect pest control programs often use periods of insecticide treatment with intermittent breaks, to prevent fixing of mutations conferring insecticide resistance. Such mutations are typically costly in an insecticide-free environment, and their frequency is determined by the balance between insecticide treatment and cost of resistance. Ace, a key gene in neuronal signaling, is a prominent target of many insecticides and across several species, three amino acid replacements (I161V, G265A, and F330Y) provide resistance against several insecticides. Because temperature disturbs neuronal signaling homeostasis, we reasoned that the cost of insecticide resistance could be modulated by ambient temperature.Experimental evolution of a natural Drosophila simulans population at hot and cold temperature regimes uncovered a surprisingly strong effect of ambient temperature. In the cold temperature regime, the resistance mutations were strongly counter selected (s = - 0.055), but in a hot environment, the fitness costs of resistance mutations were reduced by almost 50% (s = - 0.031). We attribute this unexpected observation to the advantage of the reduced enzymatic activity of resistance mutations in hot environments.We show that fitness costs of insecticide resistance genes are temperature-dependent and suggest that the duration of insecticide-free periods need to be adjusted for different climatic regions to reflect these costs. We suggest that such environment-dependent fitness effects may be more common than previously assumed and pose a major challenge for modeling climate change.