Nodupetide (1), a new cyclodepsipeptide unique in its incorporation of a 3-hydroxy-4-methylhexanoic acid (HMHA) derived motif, was discovered from Nodulisporium sp. IFB-A163, a fungus residing in the insect (Riptortus pedestris) gut. The nodupetide structure was elucidated by its MS/MS and 2D NMR spectra, and its absolute configuration by the X-ray crystallography and modified Marfey's method. Nodupetide is insecticidal against rice brown planthopper (Nilaparvata lugens) with an LD50 value of 70 ng/larva, and inhibitory towards the drug-resistant human pathogenic bacterium Pseudomonas aeruginosa with its MIC value (5.0 lM) comparable to that (3.2 lM) of ciprofloxacin, a prescribed antibacterial agent co-assayed equally.