Temperate insects spend over half their lives overwintering, weathering sub-zero temperatures, low resources, and hypoxia if they overwinter underground. To overcome these challenges in advance of winter, many insects enter diapause, a preprogrammed state of dormancy. Diapausing insects stop development, increase stress tolerance, and suppress metabolism. We only have a broad understanding of the physiological changes that occur during diapause in insects. Our aim was to elucidate the mechanisms driving diapause in the Colorado potato beetle. Beetles increase stress tolerance and suppress their metabolism by ~90% during diapause, however we do not know which specific molecules and biological processes drive these changes. Thus, our main objective was to investigate the molecular mechanisms underlying stress tolerance and metabolic suppression during diapause. We hypothesized that there would be tissue-specific shifts in the transcriptome of beetles during diapause that are consistent with stress tolerance and metabolic suppression. We used an -omics to generate specific hypotheses, some of which we then tested using physiological experiments and RNAi knockdown. Using RNA-seq, we identified expressed transcripts in tissues of diapausing beetles that encode chaperone proteins, and proteins involved in protection and repair from oxidative stress, suggesting that cytoprotection is integral for acquiring stress tolerance during diapause. We also identified expressed transcripts involved in mitophagy-driven metabolic suppression and showed that preventing mitophagy through RNA interference knockdown in diapausing beetles can reverse metabolic suppression. Our study is the first investigation of gene expression during diapause in the Colorado potato beetle using both an ‘omics and functional hypothesis testing approach.