After 25 years of genetically modified cotton cultivation in Mexico, gene flow between transgenic individuals and their wild relatives represents an opportunity for analysing the impacts of the presence of novel genes in ecological and evolutionary processes in natural conditions. We show comprehensive empirical evidence on the physiological, metabolic, and ecological effects of transgene introgression in wild cotton, Gossypium hirsutum. We report that the expression of both the cry and cp4-epsps genes in wild cotton under natural conditions altered extrafloral nectar inducibility and thus, its association with different ant species: the dominance of the defensive species Camponotus planatus in Bt plants, the presence of cp4-epsps without defence role of Monomorium ebeninum ants, and of the invasive species Paratrechina longicornis in wild plants without transgenes. Moreover, we found an increase in herbivore damage to cp4-epsps plants. Our results reveal the influence of transgene expression on native ecological interactions. These findings can be useful in the design of risk assessment methodologies for genetically modified organisms and the in situ conservation of G. hirsutum metapopulations.