Many taxa secrete chemicals to avoid predation. The Japanese blue-lined octopus Hapalochlaena cf. fasciata has high levels of potent lethal tetrodotoxin (TTX) in the muscles and skin; thus, it has been hypothesized that TTX is a defense mechanism. However, this hypothesis is based on the relationship between the location and level of TTX possession, and it has not been verified whether TTX is actually secreted in response to predators. In determining whether the external secretion of chemicals is a predator avoidance behavior, TTX must be verified as targeted to predators. In this study, TTX concentrations in the arms (muscle and skin) of octopus decreased after 3 days of predator (moray eel) presentation. In addition, TTX was only secreted in the mucus on the body surface of the octopus in the presence of a predator. Our findings showed that octopuses secrete TTX in the muscle and skin for defense, indicating that H. cf. fasciata does not necessarily require a physical contact attack by the predator to stimulate TTX secretion and can recognize predators by visual or olfactory stimuli, secreting TTX in response.