As a part of structure–activity relationship studies to elucidate structure requirements for eliciting antifungal activity, an artificial analog of amphidinol 3 (AM3) was designed. A truncated analog corresponding to the C21–C39/C52–C67 section was synthesized by using Suzuki–Miyaura coupling and Julia–Kocienski olefination as key steps. An expeditious and versatile method to construct the C53–C67 polyene section was also developed based on a linchpin strategy via successive cross-coupling reactions. The analog elicited no antifungal activity, suggesting that the two tetrahydropyran rings of AM3 are necessary to elicit antifungal activity.