Upon freezing temperatures, most insects should avoid cellular freezing by migration to warm hibernating sites, by becoming cold-hardy or by undergoing diapause development. However, a highly threatened butterfly, Parnassius bremeri, terminates egg diapause at early winter season and grows during entire winter and spring. Thus, the cold hardiness of P. bremeri needs to be explored to understand its cold tolerance limit and physiological factors. Supercooling points (SCPs) of P. bremeri vary from -10℃ to -48℃ among season. Especially, the young larvae during Jan – Mar kept SCPs at below -20℃. Larval plasma contained high level of glycerol (39.7 mM) at March, but it decreased the level (2.4 mM) at May. Transcriptome analysis indicated high levels of gene expressions associated with glycerol synthesis. Temporal expression patterns of polyol synthesis genes supported the change of glycerol. This study suggests that glycerol is a major cryoprotectant of P. bremeri to be cold-handy against freezing temperatures during winter.