Enterobacter cloacaeproduces insecticidal proteins capable of causing toxicity in pests, but the insecticidal mechanisms of these proteins for insect control remain unclear. To elucidate the mechanisms, the purified insecticidal protein fromE. cloacaeNK was administered toGalleria mellonellalarvae either by intraperitoneal injection or by feeding. The number of hemocytes, apoptosis in immune cells, and polyphenol oxidase (PO) activity ofG. mellonellalarvae were detected by hemocytometer, Annexin V-FITC/PI, and UV–vis spectrophotometer, respectively. With the extension of the invasion time of NK insecticidal protein, the number of hemocytes inG. mellonellalarvae decreased significantly (p< 0.05), whereas the apoptosis rate of hemocytes increased. The activity of PO showed a trend of rising-peak-sharp decline and the melanization reaction was deepened simultaneously. Moreover, the phagocytosis and coating capabilities of hemocytes decreased, and the intraperitoneal injection method was more effective than the feeding method. Taking together, the insecticidal protein ofE. cloacaeNK inhibits and destroys the cellular immune response ofG. mellonellalarvae, which suggests an important role in killing the host insect.