[Objectives]  To explore the effects of cold acclimation on the cold hardiness of overwintering Dendrolimus tabulaeformis larvae. [Methods]  Changes in supercooling point and major cold hardiness chemicals were measured. A thermocouple was used to measure supercooling points. Moisture, lipid, glycogen and small molecule sugar and alcohol content of larvae were determined using the delta rule, the chloroform-methanol method, phenol-sulfuric acid method and capillary gas chromatography, respectively. [Results]  The moisture content, supercooling point and lipid content of D. tabulaeformis larvae acclimated to within 5℃ below ambient temperature declined significantly, but increased in larvae acclimated to within 10℃ below ambient temperature. Glycogen content significantly increased in September, but decreased slightly in January and March after cold acclimation. Changes in small molecular sugar and alcohol content were not significant. Trehalose content slightly decreased and glycerol, glucose and galactose decreased in larvae acclimated to within 5℃ below ambient temperature, but increased when larvae were acclimated to temperatures 10℃ below ambient temperature. [Conclusion]  The observed changes in moisture, lipid, glycogen content and small molecular sugar and alcohols suggest that cold acclimation can increase the cold hardiness of D. tabulaeformis. The supercooling ability of D. tabulaeformis appears to be affected by both temperature and the time of cold acclimation, which suggests that there are optimal acclimation conditions that can maximize cold hardiness. These results provide a theoretical basis for revealing the cold hardiness mechanism and potential distribution of D. tabulaeformis.