The present study evaluated differences in the tetrodotoxin (TTX)/saxitoxins (STXs) selectivity between marine and freshwater pufferfish by performing in vivo and in vitro experiments. In the in vivo experiment, artificially reared nontoxic euryhaline freshwater pufferfish Dichotomyctere fluviatilis were intrarectally administered a mixture of TTX (24 nmol/fish) and STX (20 nmol/fish). The amount of toxin in the intestine, liver, muscle, gonads, and skin was quantified at 24, 48, and 72 h. STX was detected in the intestine over a long period of time, with some (2.7–6.1% of the given dose) being absorbed into the body and temporarily located in the liver. Very little TTX was retained in the body. In the in vitro experiments, slices of the intestine, liver, and skin tissue prepared from artificially reared nontoxic D. fluviatilis and the marine pufferfish Takifugu rubripes were incubated in buffer containing TTX and STXs (20 nmol/mL each) for up to 24 or 72 h, and the amount of toxin taken up in the tissue was quantified over time. In contrast to T. rubripes, the intestine, liver, and skin tissues of D. fluviatilis selectively took up only STXs. These findings indicate that the TTX/STXs selectivity differs between freshwater and marine pufferfish.