Isolation and structure elucidation of divergolides from Streptomyces sp. HKI0576 revealed unusual ansamycin diversification reactions and the biosynthetic flexibility of the divergolide family. The production of divergolide E in Streptomyces sp. W112 was previously increased by overexpression of div8, which belongs to ATP-binding regulators of the LuxR family. In this study, we have further characterized the products of the div8-overexpressed mutant and five new divergolide congeners (1–5) were elucidated. Among them, 1–3 features conserved divergolide A skeleton, and 4 and 5 are lactone isomers and seco variant of divergolides E and D, respectively. Divergolides O–S (1–5) showed almost no toxicity to tested human cancer cell lines of MDA-MB-231, PC3 and HeLa at 50 μM. Whereas, 4 and 5 significantly inhibited the secretion of SPI-1 effectors.