Methoprene, a juvenile hormone analog, is used to suppress populations of mosquito by inducing mortality during metamorphosis. We investigated the mode of action of methoprene in the yellow fever mosquito, Aedes aegypti . Methoprene interfered with metamorphosis, specifically affecting replacement of larval tissues. Whole mount DNA staining of midgut tissues collected from fourth larval instar and pupal stages showed that the midgut replacement started from the posterior region and progressed anteriorly. Nuclear (DAPI) and cytosolic (Phalloidin) staining of cross-sections (5 mm) of midgut tissues confirmed these observations. Application of methoprene (0.05 mg/ml) during the fourth instar interfered with programmed cell death (PCD) of larval midgut cells and proliferation, differentiation and assembly of regenerative cells into pupal midgut. Methoprene treated larvae that undergone metamorphosis contained larval midguts up to 12 hours after pupation. Quantitative real time PCR determination of mRNA levels of genes involved in PCD showed that methoprene interfered with the expression of these genes. The mode of action of methoprene appears to involve both, preventing the removal of larval tissues and the assembly of pupal tissues.