To elucidate the function of type II ice structuring protein (ISP II) in the cold tolerance and resistance ofRuditapes philippinarum, we investigatedISP IImRNA expression, the antifreeze effect of recombinant ISP II protein, and adaptation ofR. philippinarumafter acute and chronic cold stress. Bacterial freeze-thaw experiments showed thatISP IIsignificantly increased thesurvival rateofEscherichia coliBL21 (DE3) under low temperature stress, suggesting that it had a potential antifreeze effect. Furthermore, rISP II protein injections significantly increased thesurvival rateofR. philippinarumcompared with a PBS control group. In addition,succinate dehydrogenase(SDH),lactate dehydrogenase(LDH), andglutathione peroxidase(GSH-Px) activities, Malondialdehyde (MDA) content, total antioxidant capacity (T-AOC), and immune-related genes (Syk,SCD, andSOD) were measured in three populations of clams (northern, southern, and zebra). The results indicated thatISP IIwas likely to improve the cold resistance and performance ofR. philippinarumunder cold stress. These results not only provided molecular insights for improving the low-temperature resistance and immunity of shellfish but also provided useful information for improving the low temperature tolerance ofR. philippinarumin aquaculture environments.