All major insecticidal genes of Bacillus thuringiensis var. israelensis (Bti) are controlled by the sporulation-specific sigma factor Sigma E (sigE), while sigE is negatively regulated by Sigma K (sigK). Therefore, knocking out sigK plays an important role in regulating the expression of insecticidal genes in Bti. A sigK deletion mutant of B. thuringiensis var. israelensis strain Bt-59, Bt59(ΔsigK), was constructed by homologous recombination and characterized. The sigK deletion resulted in no mature spores and delayed mother cell lysis from T25 to T60, while the genetically complemented strain, Bt59(HFsigK), had mother cell lysis at T25. Compared to Bt-59, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the expression of Cry4Aa2/4Ba1 and Cyt1Aa1 proteins in Bt59(ΔsigK) increased approximately 1.67 and 1.21 times, respectively. However, there was no significant change in Cry11Aa1 protein expression between the two strains. Bioassay results showed that the sigK deletion mutation slightly reduced the insecticidal activity of Bt-59 against Culex pipiens pallens and did not obviously affect activity against Aedes albopictus.