Cold acclimation is one of the important factors in temperature adaptation for insects needing to make rapid adjustment to the seasonal temperature changes in their living environment. In a fruit fly species, Drosophila albomicans, which has a tropical origin and currently has a wide geographic distribution extended into Asian temperate regions, cold tolerance in terms of survival time at 1°C of adult flies reared at 25°C was substantially improved by a cold acclimation at 20°C for several days. Examining 29 isofemale lines from widely distributed natural populations, we observed a substantial variation in their acclimation response. However, the acclimation response was not necessarily stronger in the strains from the recently colonized temperate regions. A significantly stronger acclimation response was detected in male flies of the temperate strains when compared to those of the tropical strains. D. albomicans also showed stronger cold tolerance compared to its closely related species belonging to the D. nasuta subgroup. Among these strains, we detected a strong positive correlation between the cold tolerance change and the metabolic rate change upon the cold acclimation, suggesting their strong physiological association regulated by common genetic factors, which may have been the target of natural selection for the temperature adaptation. The response to deacclimation and reacclimation suggested that a systematic change in gene expressions is the main molecular mechanism for the cold acclimation to have effects on the cold tolerance and metabolic rate changes.