Identification of new tetrodotoxin (TTX, 1) analogues would be significant in the elucidation of its biosynthetic pathway and a study of its structure-activity relationships. In this study, a new TTX analogue, 6-deoxyTTX (2), was isolated from the ovary of the pufferfish, Takifugu pardalis, and the structure was determined using spectroscopic methods. Compound 2 was also identified in other marine animals, Nassarius snail and blue-ringed octopuses, using LC-MS. Furthermore, we investigated the voltage-gated sodium channel blocking activity of 2 by examination of the inhibitory activities to cytotoxicity induced by ouabain and veratridine in mouse neuroblastoma cells (Neuro-2a). The activities were then compared with those of 1, 11-deoxyTTX (3), and 6,11-dideoxyTTX (4). The EC50 value for 2 was estimated to be 6.5±2.2 nM, approximately 3-fold larger than that of 1 (2.1±0.6 nM) and approximately 20-fold smaller than that of 3. These results suggested that contribution of the C-6 hydroxy group to the activity is less than that of the C-11 hydroxy group.