Roselipin 1A, a bioactive natural glycolipid isolated from marine fungal metabolites, presents an unresolved configuration of its nine stereogenic centers within the polyketide chain. Herein, we elucidate the comprehensive stereostructure of roselipin 1A through an integrative approach combining predictive rule-guided analysis with synthetic chemistry. The efficient total synthesis facilitated the unequivocal confirmation of the hypothesized stereochemistry for roselipin 1A, thereby establishing its precise molecular configuration.