Gambieric acid A (1) is a marine polycyclic ether natural product that was isolated from the ciguatera causative dinoflagellate Gambierdiscus toxicus. Despite its structural similarity to ciguatoxins and brevetoxins, 1 only weakly binds to voltage-gated sodium channels and does not exhibit lethal toxicity against mice at 1 mg/kg (ip). Instead, 1 displays extraordinary potent antifungal activity against Aspergillus niger. Our previous studies on the synthesis and NMR analysis of suitably designed model compounds have strongly suggested that the stereochemical assignment of the originally proposed structure of 1 is questionable and led us to propose that the absolute configuration of the polycyclic ether domain of 1 is opposite to that of the natural product. Here, we disclose the first total synthesis and complete stereostructure of 1. The B-ring was first synthesized based on our methodology for the synthesis of medium-sized cyclic ethers. The A-ring was next forged via stereoselective bromoetherification. Suzuki-Miyaura coupling of the A/B-ring exo-olefin with an acetate-derived enol phosphate followed by ring-closing metathesis (RCM) constructed the D-ring, and mixed-thioacetalization and one-pot oxidation/methylation methodologies were used to close the C-ring. The A/BCD- and F'GHIJ-ring fragments were coupled by means of Suzuki-Miyaura coupling. After establishment of the C25 stereogenic center by exploiting the conformational bias of the F'-ring, oxidative cleavage of the F'-ring followed by elaboration of the E-ring via stereoselective allylation of a mixed thioacetal by using glycosylation chemistry, and ensuing closure of the F-ring furnished the entire polycyclic ether backbone. Finally, the J-ring side chain was introduced by means of modified Julia-Kocienski olefination to complete the first total synthesis of gambieric acid A (1). The spectroscopic data, optical rotation value, and antifungal activity of synthetic 1 matched those of the natural product. Thus, our total synthesis confirmed the correctness of our revised structure and unambiguously established the complete stereostructure of 1.