Three new 10-membered macrolides, saccharothriolides D-F (1-3), were isolated from a rare actinomycete, Saccharothrix sp. A1506. The planar structures were determined from analysis of extensive NMR and HR-ESI-MS data, and the absolute configurations were established by ECD spectroscopy analysis. Saccharothriolides D (1) and E (2) were determined to be C-2 epimers of saccharothriolides A (4) and B (5), respectively. Saccharothriolide F (3) was identified to be a demethylated congener of saccharothriolides D (1) and A (4) at the C-2 position. The availability of compounds 1-6 enabled a structure-activity relationship study that revealed the importance of the phenolic hydroxy group at C-2″ and the stereochemistry of C-2 for the inhibition of human fibrosarcoma HT1080 cell growth.