Abstract: Obscure puffer (Takifugu obscurus) is a euryhaline fugu species and belongs to the Family Tetraodontidae of teleost fish. In China, the Takifugu species is very popular because of its delicious taste and high nutrition, and it is widely farmed on a commercial scale. In recent years, the aquaculture area for obscure puffer is increasing year by year, however, the industry is confronted with many challenges, such as outbreak of diseases and overwintering culture. When winter comes, the growth and breeding of obscure puffer are affected by the environmental temperature. Furthermore, the dramatic decrease of water temperature in winter will cause mass mortality of obscure puffer, and then huge economic losses. Therefore, it is an urgent need for the further development of the industry to obtain low-temperature tolerance fish species through artificial breeding methods, and the premise of this work is to analyze the low-temperature tolerance mechanism of obscure puffer. To explore the response mechanism of obscure puffer to low temperature environment, we cloned the cDNA sequences of tolerance related genes CIRBP, HMGB1 and AFP-Ⅳ and then characterized their molecular features and potential functions. Real-time PCR analysis showed that CIRBP and HMGB1 were highly expressed in the hypothalamus, liver and muscle, while AFP-Ⅳ was mainly expressed in the liver. The obscure puffer was subjected to acute cold stress, then tissue samples were taken at different time periods. Real-time PCR analysis showed that the expression patterns of the above three genes were different in liver and hypothalamus. For example, the expression of CIRBP increased significantly at 48h in liver, while only slightly increased at 12h and 48h in hypothalamus. The expression of HMGB1 showed a gradual increase and reached the highest level at 48h in liver, while it first increased and then decreased in hypothalamus tissue. It reached the maximum at 2h after treatment, decreased at 2—8h, and reached the lowest at 8h, then recovered to the initial level. The expression of AFP-Ⅳ in liver showed no significant change from 0 to 24h, but increased to the maximum at 48h. The antifreeze role of AFP-Ⅳ was further investigated by heterologous expression in Escherichia coli. It was found that the expression of AFP-Ⅳ in E. coli exerted antifreeze effect at –80℃, and the antifreeze activity became apparent with the increase of the concentration of AFP-Ⅳ. Collectively, the results showed that the three genes were involved in the response to low temperature stress, which laid a foundation for further exploring the mechanism of low temperature tolerance in obscure puffer.