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The binary toxin ‘ Photorhabdus insect-related’ proteins (PirAB) produced by Photorhabdus luminescens have been reported to possess both injectable and oral activities against a range of insects. Here, PirAB-fusion protein was constructed by linking pirA and pirB genes with the flexible linker (Gly4Ser)3 DNA encoding sequence and then efficiently expressed in Escherichia coli . To better understand the role of PirAB toxin played in the process of invasion, its cytotoxicity against insect midgut CF-203 cells was investigated. Application of purified PirAB-fusion protein as well as PirA/PirB mixture caused loss of viability of CF-203 cells after 24 h incubation. CF-203 cells treated by PirAB-fusion protein displayed morphological changes typical of apoptosis, such as cell shrinkage, cell membrane blebbing, nuclear condensation and DNA fragmentation. Moreover, PirAB-fusion protein also exhibited injectable insecticidal activity against Spodoptera exigua larvae. The bodies of S. exigua fourth-instar larvae injected with PirAB-fusion protein turned completely black. Thus, we concluded that PirAB-fusion protein possessed similar biological activity (cytotoxicity and insecticidal activity) to PirA/PirB mixture, which would enable it to be used as an efficient agent for pest control.