<jats:title>Summary</jats:title><jats:p>Hopanoids are bacterial surrogates of eukaryotic membrane sterols and among earth's most abundant natural products. Their molecular fossils remain in sediments spanning more than a billion years. However, hopanoid metabolism and function are not fully understood. <jats:styled-content style="fixed-case"><jats:italic>B</jats:italic></jats:styled-content><jats:italic>urkholderia</jats:italic> species are environmental opportunistic pathogens that produce hopanoids and also occupy diverse ecological niches. We investigated hopanoids biosynthesis in <jats:styled-content style="fixed-case"><jats:italic>B</jats:italic></jats:styled-content><jats:italic>urkholderia cenocepacia</jats:italic> by deletion mutagenesis and structural characterization of the hopanoids produced by the mutants. The enzymes encoded by <jats:styled-content style="fixed-case"><jats:italic>hpnH</jats:italic></jats:styled-content> and <jats:styled-content style="fixed-case"><jats:italic>hpnG</jats:italic></jats:styled-content> were essential for production of all <jats:styled-content style="fixed-case">C</jats:styled-content><jats:sub>35</jats:sub> extended hopanoids, including bacteriohopanetetrol (<jats:styled-content style="fixed-case">BHT</jats:styled-content>), <jats:styled-content style="fixed-case">BHT</jats:styled-content> glucosamine and <jats:styled-content style="fixed-case">BHT</jats:styled-content> cyclitol ether. Deletion of <jats:styled-content style="fixed-case"><jats:italic>hpnI</jats:italic></jats:styled-content> resulted in <jats:styled-content style="fixed-case">BHT</jats:styled-content> production, while Δ<jats:styled-content style="fixed-case"><jats:italic>hpnJ</jats:italic></jats:styled-content> produced only <jats:styled-content style="fixed-case">BHT</jats:styled-content> glucosamine. Thus, <jats:styled-content style="fixed-case">HpnI</jats:styled-content> is required for <jats:styled-content style="fixed-case">BHT</jats:styled-content> glucosamine production while <jats:styled-content style="fixed-case">HpnJ</jats:styled-content> is responsible for its conversion to the cyclitol ether. The Δ<jats:styled-content style="fixed-case"><jats:italic>hpnH</jats:italic></jats:styled-content> and Δ<jats:styled-content style="fixed-case"><jats:italic>hpnG</jats:italic></jats:styled-content> mutants could not grow under any stress condition tested, whereas Δ<jats:styled-content style="fixed-case"><jats:italic>hpnI</jats:italic></jats:styled-content>, Δ<jats:styled-content style="fixed-case"><jats:italic>hpnJ</jats:italic></jats:styled-content> and Δ<jats:styled-content style="fixed-case"><jats:italic>hpnK</jats:italic></jats:styled-content> displayed wild‐type growth rates when exposed to detergent, but varying levels of sensitivity to low <jats:styled-content style="fixed-case">pH</jats:styled-content> and polymyxin <jats:styled-content style="fixed-case">B</jats:styled-content>. This study not only elucidates the biosynthetic pathway of hopanoids in <jats:styled-content style="fixed-case"><jats:italic>B</jats:italic></jats:styled-content><jats:italic>. cenocepacia</jats:italic>, but also uncovers a biosynthetic role for the conserved proteins <jats:styled-content style="fixed-case">HpnI</jats:styled-content>, <jats:styled-content style="fixed-case">HpnJ</jats:styled-content> and <jats:styled-content style="fixed-case">HpnK</jats:styled-content> in other hopanoid‐producing bacteria.