Tetrodotoxin, a toxic principle of puffer fish intoxication, is one of the most famous marine natural products owing to its complex structure and potent biological activity, which leads to fatal poisoning. Continuous synthetic studies on tetrodotoxin and its analogues to elucidate biologically interesting issues associated with tetrodotoxin have led to the development of versatile routes for a variety of tetrodotoxin derivatives. With the aim of investigating the structure-activity relationship of tetrodotoxin with voltage-gated sodium channels, this study describes the first total syntheses of 5-deoxytetrodotoxin, a natural analogue of tetrodotoxin, and 8-deoxytetrodotoxin, an unnatural analogue, from a newly designed, versatile intermediate in an efficient manner. An estimation of the biological activities of these compounds reveals the importance of the hydroxy groups at the C-5 and C-8 positions on the inhibition of voltage-gated sodium channels.