The tobacco caterpillar, Spodoptera litura F., is one of the most devastating, cosmopolitan polyphagous pests affecting major crops that significantly impact agricultural productivity. The present study aimed to evaluate the toxicological effect of seed extract of the medicinal plant, Annona squamosa L. and the histological effect on the midgut of Spodoptera litura F., under laboratory conditions. The crude methanolic extract of A. squamosa seed was tested against the third instar larvae of S. litura at five different concentrations viz., 0.5%, 1.0%, 1.5%, 2.0% and 2.5 % by leaf dip bioassay method. The seed extract exhibited larval mortality of 96.67%, 83.37 %,70%, 53.33% and 40 % at the concentrations 2.5 %, 2.0%, 1.5%, 1.0% and 0.5%, respectively.The results indicated that the response of larval mortality to the seed extract was dose-dependent. The dose of 0.5% showed the lowest mortality (40.00%), while the dose of 2.5 % showed maximum larval mortality (96.67%). Hence they were subjected to histological analysis. The anatomical sectioning of S. litura larval midgut treated with 0.5% concentration showed disruption in the peritrophic membrane and striated border of epithelial cells. The midgut of larvae treated with 2.5 % concentration showed an irregular epithelium and high vacuolization in the cytoplasmic cells. The results indicated that methanol extract of A. squamosa seed extract has the ability to cause changes in the midgut region, thereby affecting the digestion and nutrient absorption of S. litura larvae, which will lead to a debilitating effect on the development of larvae. Based on the present study A. squamosa seed extract will be a potential biopesticide for managing S. litura.