Cold acclimation is a critical physiological adaptation for coping with seasonal cold. By increasing their cold tolerance individuals can remain active for longer at the onset of winter and can recover more quickly from a cold shock. In insects, despite many physiological studies, little is known about the genetic basis of cold acclimation. Recently, transcriptomic analyses in Drosophila virilis and D. montana revealed candidate genes for cold acclimation by identifying genes upregulated during exposure to cold. Here, we test the role of myo-inositol-1-phosphate synthase (Inos), in cold tolerance in D. montana using an RNAi approach. D. montana has a circumpolar distribution and overwinters as an adult in northern latitudes with extreme cold. We assessed cold tolerance of dsRNA knock-down flies using two metrics: chill-coma recovery time (CCRT) and mortality rate after cold acclimation. Injection of dsRNAInos did not alter CCRT, either overall or in interaction with the cold treatment, however it did induced cold-specific mortality, with high levels of mortality observed in injected flies acclimated at 5°C but not at 19°C. Overall, injection with dsRNAInos induced a temperature-sensitive mortality rate of over 60% in this normally cold-tolerant species. qPCR analysis confirmed that dsRNA injection successfully reduced gene expression of Inos. Thus, our results demonstrate the involvement of Inos in increasing cold tolerance in D. montana. The potential mechanisms involved by which Inos increases cold tolerance are also discussed.