The mechanisms of cold tolerance in insects which are exposed to suboptimal temperatures for survival include accumulation of cryoprotectants and expression of heat shock protein genes (hsps). Spodoptera exigua is a freeze avoidance insect, which can overwinter as third instar larvae in China. Our previous study illustrated that seasonal and diel fluctuating temperatures enhanced its cold hardening. The survival mechanism for this stage, however, is poorly understood. We hypothesized that the survival mechanisms in third instar larvae of S. exigua were correlated with the induction of cryoprotectants and/or expression of hsps. Results showed that trehalose as the main cryoprotectant was accumulated during the cold acclimation (CA), and the level of accumulation fell off sharply when the CA treatment was discontinued (discontinued cold acclimation, DCA). Furthermore, inducible hsp90 was down-regulated in all treatments compared with the control, and hsp70 was up-regulated under conditions of CA and RCH (rapid cold hardening), which induced 1.23- and 44.00-fold increases compared with the control. Therefore, we considered that both trehalose (as the main cryoprotectant) and hsps (i.e. hsp90 and hsp70) were involved in the regulation of physiological activity of third instar larvae of S. exigua to resist low temperatures during winter. These results are helpful for us to understand the mechanisms underlying this species' ecological adaptability and stress tolerance.