Tetrodotoxin, a toxic principle of puffer fish poisoning, is one of the most famous marine natural products because of the complex structure and its potent biological activity. Since the structure elucidation in 1964, the toxin has been recognized as a formidable target molecule for total synthesis. We achieved the first asymmetric total synthesis commencing from 2-acetoxy-tri-O-acetyl-D-glucal as a chiral starting material. Highly hydroxylated cyclohexane was constructed by intramolecular aldol condensation of a precursor having methyl ketone with dihydroxyacetone, which was synthesized through Sonogashira coupling, Claisen rearrangement and regioselective hydroxylations of acetone moiety. Since installation of nitrogen functionality by the attempted Overman rearrangement was unsuccessful, we employed intramolecular conjugate addition of carbamate to unsaturated ester for the purpose. The α-hydroxyl lactone moiety was synthesized by epoxide opening with enolate of aldehyde followed by oxidation-reduction of the resulting cyclic vinylether. The lactone was then protected as ortho ester, and guanidinylation followed by cleavage of the 1, 2-glycol gave a fully protected tetrodotoxin. Protective group elaboration furnished the total synthesis of tetrodotoxin in an enantiomerically pure form.