1. Adaptive responses to thermal stress typically involve a range of plastic acclimatory responses in ectothermic animals. The mechanisms underlying phenotypic plasticity in inducible cold tolerance are complex and not fully understood. 2. Here we investigated how thermoperiodic cold acclimation affected the cold tolerance and the metabolome of adult Drosophila melanogaster. We have used targeted GC⁄MS metabolomic profiling to address whether cold acclimation induced specific metabolic changes and affected the dynamics of the homeostatic response following different types of cold stress (acute and chronic). 3. Developmental combined with gradual adult acclimation strongly promoted cold tolerance. This phenotypic variation was associated with significant metabolic changes, among which some sugars, polyamines and metabolic intermediates are fingerprints of these changes. Cold acclimation allowed individuals to maintain metabolic homeostasis, whereas non-acclimated counterparts suffered from deep and persistent homeostatic perturbations. 4. This study gives a fertile ground for future research in disentangling the role of several metabolites putatively involved in cold acclimation and cold stress response. It also provides insight into the mechanisms by which cold acclimation is achieved in D. melanogaster and gives a basis for elucidating the evolution of plastic responses to thermal variations.