Abstract Xenorhabdus nematophila, a Gram-negative proteobacterium belonging to the family Enterobacteriaceae and associated symbiotically with soil entomopathogenic nematodes, Steinernema carpocapsae, is pathogenic to a wide range of insects. A protein complex with insecticidal activity was isolated from the cells of X. nematophila HB310 strain using methods of salting out and native polyacrylamide gel electrophoresis (PAGE). Seven polypeptides ranging 50∼250 kDa were well separated from the protein complex (named Xnpt) by sodium dodecyl sulfate (SDS)-PAGE, five of which are identified as XptA2, xptC1, XptB1, GroEL and hypothetical protein by matrix-assisted laser desorption-time-of-flight mass spectrometry (MALDI-TOFMS). Xnpt showed high oral virulence to larvae of diamondback moth (DBM), Plutella xylostella L. (Lepidoptera, Plutellidae) as its median lethal concentration (LC50) against second and third instar larvae were 331.45 ng/mL and 553.59 ng/mL at 72 h, respectively. The histological analysis of Xnpt-fed DBM larvae showed extensive histopathological effects on the midgut. Biochemical analysis indicated that Xnpt markedly inhibited the activities of three important enzymes in the midgut. Overall, our data showed that the protein complex isolated from X. nematophila HB310 induced the antifeedant and death of insects by destroying midgut tissues and inhibiting midgut proteases activities.