Abstract <p><i>Bacillus thuringiensis</i> (Bt)–based microbial insecticides are crucial for controlling the maize pest, <i>Spodoptera frugiperda</i> (Smith) (Lepidoptera: Noctuidae). While the insecticidal efficacy of Bt has been well-documented, its sublethal cytogenetic toxicity on midgut cells remains less explored. This study aimed to evaluate the sublethal concentration toxicity of <i>Bacillus thuringiensis</i>, var. <i>Kurstaki</i> (Btk), on survival percentage, genotoxicity, histopathology, and ultrastructure of the midgut cells of <i>S. frugiperda</i> larvae. By calculating lethal concentration (LC) values (LC₂₅, LC₅₀, LC₇₅, and LC₉₀) in the laboratory, the insecticidal efficacy was ascertained. The Btk lethal concentration was toxic to third-instar <i>S. frugiperda</i> larvae after 48&#xa0;h, with an LC₅₀ of 109&#xa0;µg/100&#xa0;mL (106–112&#xa0;µg/100&#xa0;mL). Subsequently, the results demonstrated that LC₂₅ resulted in a 22% reduction in larval survival percentages, so larval midgut tissues treated with the sublethal concentration (LC₂₅) were isolated for further cytogenetic analysis. Consequently, Btk treatment markedly disrupted midgut cells, inducing micronuclei (MN) formation and reducing <i>SfDnmt2</i> mRNA gene expression considerably. Furthermore, cytotoxicity has been shown in <i>S. frugiperda</i> midgut epithelial cells by cellular disorganization, fragmentation and protrusion, peritrophic matrix rupture, and microvilli degeneration. Btk sublethal concentration also induced cytotoxicity by causing apoptosis and necrosis in <i>S. frugiperda</i> midgut cells. These findings suggest that sublethal concentrations of Bt induce significant cytogenetic stress, which may complement the role of Bt-based biopesticides in integrated pest management strategies as a promising, environmentally safe method for decreasing the survival rate and controlling the pest <i>S. frugiperda</i>.</p> Graphical Abstract <p></p>