Pyrrolizidine alkaloids (PAs) are widespread plant natural products with potent toxicity and bioactivity. Herein, the identification of bacterial PAs from entomopathogenic bacteria using differential analysis by 2D NMR spectroscopy (DANS) and mass spectrometry is described. Their biosynthesis was elucidated to involve a non-ribosomal peptide synthetase. The occurrence of these biosynthesis gene clusters in Gram-negative and Gram-positive bacteria indicates an important biological function in bacteria. The structures of bacterial pyrrolizidine alkaloids from Xenorhabdus, the pyrrolizixenamides, have been elucidated by two-dimensional NMR spectroscopy and mass spectrometry. Their biosynthesis is shown to involve the bimodular non-ribosomal peptide synthetase PxaA and the unique monooxygenase PxaB. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.