The present study investigated the intra-vitam larval migration of Anisakis pegreffii, which is naturally infected, from the intestine to the viscera and the musculature of live spotty-bellied greenling, Hexagrammos otakii, under the different seawater temperatures. The results showed that the increase in the ratio of larval migration from the intestine to the viscera correlated with the increase in the water temperature in the aquarium. The intra-vitam larval migration ratios from the intestine to the viscera were 60% (63/105) at 15°C, 1.64% (3/183) at 12°C, 1.16% (2/172) at 9°C and 1.42% (2/141) in the control group (7.5°C). The free larvae showed no, slight and strong movement at 9°C, 12°C and 15°C, respectively, in saline water, and the larvae were present in a coiled state at 9°C and 12°C. The results suggest that temperature plays an important role in the intra-vitam larval migration of A. pegreffii. In addition, the free larval behavior of A. pegreffii in vitro increased with increase in temperature. Therefore, to prevent human anisakiasis caused by the consumption of raw fish, it is important that the temperature of the aquarium must be maintained below 12°C, which is the critical temperature zone.