<jats:title>Abstract</jats:title><jats:p>The <jats:italic>ast</jats:italic> gene cluster (GenBank accession numbers KF813023.1 and KP284551) was characterized to be responsible for the biosynthesis of ansatrienins in <jats:italic>Streptomyces</jats:italic> sp. XZQH13, which contains <jats:italic>astC</jats:italic>, <jats:italic>astF1</jats:italic>, and <jats:italic>astF2</jats:italic> genes involved in the assembly of the <jats:italic>N</jats:italic>‐cyclohexanoyl <jats:sc>d</jats:sc>‐alanyl side chain and the hydroxylation of C‐19, respectively. Further to investigating the biosynthetic mechanism of ansatrienins, herein we constructed the mutant strains XZQH13OEΔ<jats:italic>astF2</jats:italic> and XZQH13OEΔ<jats:italic>astC</jats:italic>Δ<jats:italic>astF2</jats:italic>. Three new ansatrienin analogues, namely, ansatrienols I–K (<jats:bold>1</jats:bold>–<jats:bold>3</jats:bold>), along with trienomycinol (<jats:bold>4</jats:bold>) and 3‐<jats:italic>O</jats:italic>‐demethyltrienomycinol (<jats:bold>5</jats:bold>), were isolated from the XZQH13OEΔ<jats:italic>astC</jats:italic>Δ<jats:italic>astF2</jats:italic> strain, and trienomycin A (<jats:bold>6</jats:bold>) and trienomycin G (<jats:bold>7</jats:bold>) were isolated from the XZQH13OEΔ<jats:italic>astF2</jats:italic> strain. Their structures were determined by a combination of high‐resolution MS (ESI) and 1D and 2D NMR spectroscopy. Accordingly, a pathway for the biosynthesis of these new ansatrienins was proposed.