Changes in susceptibility of Spodoptera littoralis (Lepidoptera: Noctuidae) were investigated against some insecticides. Newly hatched eggs collected from “Al-Qnawiyah," in El-Behira governorate were reared on cotton leaves of the same location up to the 4^th instar in the laboratory to gain the 1^st generation of field strain (1GFS) owned habitual gut-bacteria (HGB) versus free gut-bacteria (FGB) reared on leaves treated with antibiotics. HGB revealed less susceptibility than FGB realized by LC₅₀ ratios (HGB/ FGB) of lufenuron, emamectin benzoate, chlorpyrifos and alpha-cypermethrin with 15.38, 5.08, 3.39 and 2.17 folds in, respectively. Isolated strains from the 4^th instar's gut were molecularly sequenced for 16S rRNA gene as Enterobacter cloacae Ag10, Pseudomonas aeruginosa Fs40 and Pseudomonas aeruginosa Fs30. These isolates were submitted for growth activity confirmation test for 3 days of cultivation on insecticide-based minimal medium 9 (from 20 to 500 mg L^-1) versus glucose-based medium (control). E. cloaca Ag10 grew on all concentrations of alpha-cypermethrin likewise P. aeruginosa Fs40 on lufenuron and chlorpyrifos. P. aeruginosa Fs30 did not grow on lufenuron but was limited on the others. Semi-field trials in two successive seasons of cotton crop in the foregoing site against the 4^th instar of 1GFS showed the long-term toxicity of these insecticides against FGB more than HGB. Therefore, the identified gut-bacterial strains are the hidden cause behind susceptibility shifts in S. littoralis to these insecticides.