Smooth septate junctions (sSJs) contribute to the epithelial barrier, which restricts leakage of solutes through the paracellular route in epithelial cells of the Drosophila midgut. We previously identified three sSJ-associated membrane proteins, Ssk, Mesh and Tsp2A, and showed that these proteins were required for sSJ formation and intestinal barrier function in the larval midgut. Here, we investigated the roles of sSJs in the Drosophila adult midgut. Depletion of any of the sSJ proteins from enterocytes resulted in remarkably shortened lifespan and intestinal barrier dysfunction in flies. Interestingly, the sSJ-protein-deficient flies showed intestinal hypertrophy accompanied by accumulation of morphologically abnormal enterocytes. The phenotype was associated with increased stem cell proliferation and activation of the MAPK and Jak-Stat pathways in stem cells. Loss of the cytokines Unpaired 2 and Unpaired 3, which are involved in Jak-Stat pathway activation, reduced the intestinal hypertrophy, but not the increased stem cell proliferation, in flies lacking Mesh. The present findings suggest that SJs play a crucial role in maintaining tissue homeostasis through regulation of stem cell proliferation and enterocyte behavior in the Drosophila adult midgut.