Tetrodotoxin (TTX) was intramuscularly administered to nontoxic cultured specimens of the pufferfish Takifugu rubripes to investigate differences in the toxin transfer and accumulation profiles between the skin and liver. Test fish were administered TTX at doses of 30 (Low dose; LD), 100 (Medium dose; MD), and 300 (High dose; HD) μg/individual, respectively. Liquid chromatography/mass spectrometry analysis for TTX revealed that the TTX concentration in both the skin (0.48–1.7 μg/g) and liver (0.43–6.0 μg/g) at 24 h after the toxin administration increased with an increase in the dose. The TTX accumulation ratio (ratio (%) of accumulated TTX in each tissue (μg/tissue) to the administered dose (μg/individual)) of the skin (11.1–38.6) significantly decreased with an increase in the dose, whereas that of the liver (18.4–21.3) was almost constant irrespective of the dose. Immunohistochemical observations of the skin sections revealed TTX-positive signals in basal cells in LD, but as the dose increased, TTX-positive signals were also observed in the epidermis. In the liver sections, TTX-positive signals were rarely observed in LD and MD, but the TTX was distributed throughout the liver tissue in HD.