Abstract Although puffer fish contain tetrodotoxin (TTX) at a high concentration mainly in liver, the underlying mechanism remains to be elucidated. In the present study, uptake of TTX into the liver tissue slices of puffer fish Takifugu rubripes was investigated by in vitro incubation experiment. When T. rubripes liver slices were incubated with 0–2000 μM TTX at 20 °C for 60 min, the uptake rates exhibited non-linearity, suggesting that the TTX uptake into T. rubripes liver is carrier-mediated. The TTX uptake was composed of a saturable component ( V max 47.7±5.9 pmol/min/mg protein and K m 249±47 μM) and a non-saturable component ( P dif 0.0335±0.0041 μL/min/mg protein). The uptake of TTX was significantly decreased to 0.4 and 0.6 fold by the incubation at 5 °C and the replacement of sodium-ion by choline in the buffer, respectively, while it was not affected by the presence of 1 mM l-carnitine, p-aminohippurate, taurocholate or tetraethylammonium. The TTX uptake by black scraper Thamnaconus modestus liver slices was much lower than that of T. rubripes and independent of the incubation temperature, unlike T. rubripes. These results reveal the involvement of carrier-mediated transport system in the TTX uptake by puffer fish T. rubripes liver slices.