Opantimycin A, a rare antimycin-class antibiotic without the macrolide core, was isolated from Streptomyces sp. RK88-1355 in 2017. In this study, we explored the total synthesis and stereochemical assignment of opantimycin A. The synthesis of all potential diastereomers has been accomplished via traceless Staudinger ligation. A comparison of the spectroscopic data of the synthesized compounds with that reported for the natural product confirmed that the absolute configuration of the natural product was (14S,17R,21R). Two analogous compounds were prepared, where the Dhb ((Z)-dehydrobutyrine) moiety was replaced with Dha (dehydroalanine) or ΔVal moieties, respectively. The inhibitory activities of these synthetic compounds against the production of the anti-inflammatory cytokine IL-6 were evaluated, and two potential candidates for further development as anti-inflammatory agents were identified.