<jats:title>Abstract</jats:title><jats:p><jats:italic>Pseudomonas aeruginosa</jats:italic> displays an impressive metabolic versatility, which ensures its survival in diverse environments. Reported herein is the identification of rare azetidine‐containing alkaloids from <jats:italic>P. aeruginosa</jats:italic> PAO1, termed azetidomonamides, which are derived from a conserved, quorum‐sensing regulated nonribosomal peptide synthetase (NRPS) pathway. Biosynthesis of the azetidine motif has been elucidated by gene inactivation, feeding experiments, and biochemical characterization in vitro, which involves a new <jats:italic>S</jats:italic>‐adenosylmethionine‐dependent enzyme to produce azetidine 2‐carboxylic acid as an unusual building block of NRPS. The mutants of <jats:italic>P. aeruginosa</jats:italic> unable to produce azetidomonamides had an advantage in growth at high cell density in vitro and displayed rapid virulence in <jats:italic>Galleria mellonella</jats:italic> model, inferring functional roles of azetidomonamides in the host adaptation. This work opens the avenue to study the biological functions of azetidomonamides and related compounds in pathogenic and environmental bacteria.