<jats:title>Abstract</jats:title><jats:p>A new cyclic lipopeptide, phototemtide A (<jats:bold>1</jats:bold>), was isolated from <jats:italic>Escherichia coli</jats:italic> expressing the biosynthetic gene cluster <jats:italic>pttABC</jats:italic> from <jats:italic>Photorhabdus temperata</jats:italic> Meg1. The structure of <jats:bold>1</jats:bold> was elucidated by HR‐ESI‐MS and NMR experiments. The absolute configurations of amino acids and 3‐hydroxyoctanoic acid in <jats:bold>1</jats:bold> were determined by using the advanced Marfey's method and comparison after total synthesis of <jats:bold>1</jats:bold>, respectively. Additionally, three new minor derivatives, phototemtides B–D (<jats:bold>2</jats:bold>–<jats:bold>4</jats:bold>), were identified by detailed HPLC–MS analysis. Phototemtide A (<jats:bold>1</jats:bold>) showed weak antiprotozoal activity against <jats:italic>Plasmodium falciparum</jats:italic>, with an IC<jats:sub>50</jats:sub> value of 9.8 μ<jats:sc>m</jats:sc>. The biosynthesis of phototemtides A–D (<jats:bold>1</jats:bold>–<jats:bold>4</jats:bold>) was also proposed.