The blue gum chalcid, Leptocybe invasa Fisher & La Salle (Hymenoptera: Eulophidae: Tetrastichinae) is an invasive pest in Eucalyptus plantations in the world. Low temperature is one of the most important factors influencing the invasion and colonization of L. invasa. However, effects of diel temperature fluctuations (e.g. rapid cold-hardening (RCH)) on survival of L. invasa are less well understood. Here, we investigated the survival and differentially expressed proteins of L. invasa adults' response to different pre-treatment protocols. Survival of adults exposed at -20 °C decreased from 100 % (0 min and 15 min) to 5 ± 2 % (45 min). Survival of L. invasa adults, exposed at -20 °C for 45 min, was the highest at the pre-treatment temperature of 4 °C for 90 min. Mass spectrometry analysis and database searching helped us to identify a total of 85 proteins that exhibited differential expression, in which up-regulated expression of 28 proteins and down-regulated expression of 57 proteins were observed in comparison with control. Up-regulated proteins were members of heat shock proteins and ribosomal proteins, while down-regulated proteins were members of energy metabolism and degradation. In conclusion, RCH could enhance the cold hardiness of L. invasa adults.Theadaptive responsemayallow L. invasa to invade the extreme environment in the world.