The juvenile hormone analogs (JHA) are known to disrupt insect development but the molecular mechanisms of their action have been studied only in a few model insects belonging to orders Diptera and Lepidoptera. Here, we investigated the mechanisms of JHA action in red flour beetle, Tribolium castaneum, belonging to the order Coleoptera. Application of JHA during penultimate and final instar larval stages blocked larval-pupal metamorphosis and induced supernumerary larval molts. When compared to the control insects undergoing larval-pupal molt, down-regulation of expression of transcription factor, Broad, and up-regulation of other genes involved in 20-hydroxyecdysone (20E) action (FTZ-F1, E74) were observed in JHA-treated larvae undergoing supernumerary larval molts. The presence of JHA during the final instar larval stage blocked the midgut remodeling wherein programmed cell death (PCD) of larval cells and proliferation and differentiation of imaginal cells to pupal gut epithelium were impaired. The comparative analysis of 20E-induced gene expression in the midguts of JHA-treated and control insects revealed that JHA suppressed the expression of EcRA, EcRB, Broad, E74, E75A, and E75B, resulting in a block in PCD as well as proliferation and differentiation of imaginal cells.