Abstract The role of chaperone proteins in the winter survival of insects was evaluated in freeze tolerant gall fly larvae, Eurosta solidaginis. Levels of four heat shock proteins (Hsp110, Hsp70, Hsp60, Hsp40), two glucose-regulated proteins (Grp75, Grp78) and three others (tailless complex polypeptide 1 [TCP-1], αA-crystallin, αB-crystallin) were tracked in outdoor larvae from September to April and, in addition, laboratory experiments assessed chilling, freezing, and anoxia effects on these proteins. Gall fly larvae showed consistent elevation of Hsp110, Hsp70, Hsp40, Grp78 and αB-crystallin over the late autumn and winter months, generally 1.5–2.0-fold higher than September values. This suggests that these proteins contribute to cell preservation over the winter months via protection and stabilization of macromolecules. By contrast, levels of the mitochondrial Hsp60 fell to just 40% of September values by midwinter, paralleling the responses by numerous mitochondrial enzymes and consistent with a reduction in total mitochondria numbers over the winter. None of the proteins were altered when 15 °C acclimated larvae were chilled to 3 °C for 24 h but Hsp70, Hsp40 and Grp75 increased during freezing at −16 °C for 24 h whereas others (Hsp110, TCP-1 and both crystallins) increased significantly after larvae thawed at 3 °C. Anoxia exposure (24 h under N2 gas at 15 °C) elevated levels of Hsp70, Grp78 and the two crystallins. Levels of active hyperphosphorylated heat shock transcription factor (HSF1) were also analyzed, giving an indication of the state of hsp gene transcription in the larvae. HSF1 was high in September and October but fell to less than 40% of September values in midwinter consistent with suppression of gene transcription in diapause larvae. HSF1 levels responded positively to freezing and increased robustly by 4.9-fold under anoxia. Overall, the data provide strong evidence for the importance of protein chaperones as a mechanism of cell preservation in freeze tolerant insects.