To investigate seasonal changes in the whole body content and tissue distribution of tetrodotoxin (TTX) in the pufferfish Takifugu niphobles, wild individuals were collected from the coastal waters around Miura Peninsula from November 2010 to May 2012, and their tissues were subjected to LC-MS/MS analysis. Fish that were sexually mature were classified as being in the maturation period (April), the spawning period (May–July) or the “ordinary period” (i.e., other months). In both sexes, gonad somatic index rapidly increased during the maturation period and then decreased during the spawning period. Whole body TTX content was significantly higher during the maturation/spawning period than in the ordinary period. Through all seasons, TTX was localized in the skin or ovary in females and in the skin or liver in males: the difference in TTX localization between females and males was particularly evident during the spawning period.