The bacterio-insecticide Bacillus thuringiensis subsp. israelensis (Bti) represents currently a safe alternative to chemical insecticides in mosquito-borne diseases control and is intensively used worldwide. In the French Rhône-alpine mosquito Aedes rusticus Rossi, several genomic signatures of selection were detected in populations treated for more than 15 years with Bti. In this study we investigated whether detoxifying enzyme activities such as cytochromes P450, esterases and glutathione S-transferases can play a role in Bti resistance mechanisms in these populations. To examine the effect of Bti treatment on larval resistance and detoxifying enzyme activities, Bti mortality and enzymatic activities were measured in treated and untreated sites coming from four different locations in the Rhône-Alpes region. The results revealed a significant global decrease in Bti mortality in treated populations, but showed low resistance ratio up to 7.9-fold between treated and untreated sites. A positive correlation has been observed between larval resistance to Bti and glutathione S-transferase activities, raising the question of its potential role in Bti resistance. A better understanding of all the mechanisms potentially involved in Bti resistance is necessary to insure the long-term efficiency of this bacterio-insecticide.