Acetogenins are secondary metabolites exclusively produced by Annonaceae, which have antitumor, cytotoxic, and pesticide activities. In this study, we evaluated the larvicidal and cytotoxic effect of squamocin from Annona squamosa on Aedes aegypti (Diptera: Culicidae) midgut. The compound was solubilized in 2% Tween 20 at 10, 20, 50, 80 and 100 ppm. The assay was conducted in a completely randomized design with four replications, each with 20 third-instar larvae. Larval mortality was assessed every hour until total mortality, and the data were subjected to Probit analysis. Cellular damage was evaluated every 30 min in groups comprising five larvae subjected to squamocin at 50 and 100 ppm for 240 min. The total larval mortality occurred after 360 min following application of 50, 80, and 100 ppm squamocin, and 600 min after applying other concentrations with LC50 at 6.4 ppm. Both 50 and 100 ppm of squamocin showed cytotoxic activity in the midgut epithelium of A. aegypti after 240 min with 50 ppm resulting in midgut cells with light cytoplasm containing small vacuoles, whereas at 100 ppm were found cells with cytoplasm highly vacuolated, damaged apical surface and cell protrusion toward the gut lumen. In conclusion, squamocin has the potential to control A. aegypti.