20-hydroxyecdysone (20E) plays an inhibitory role in the proliferation of Mbn2 cells. We used Mbn2 cells to study changes in gene expression in response to 20E and juvenile hormone (JH). Quantitative real time RT-PCR analyses showed that EcR, E93, Br-C, Imd, Rel, Dredd, Eip63E, Rpr, E75, and Fadd mRNA levels increased in cells exposed to 1 μM 20E for 24 hr. Exposure of these cells to JH III alone did not affect the mRNA levels of these genes. However, exposure of these cells to both 20E and JH III resulted in JH III blocking 20E induced mRNA expression. Immunocytochemical analyses using Broad and EcR antibodies confirmed the above results; higher levels of these proteins were present in cells exposed to 1 μM 20E when compared to the expression levels in cells exposed to DMSO or 1 μM each of 20E and JH III. In addition, exposure of these cells to 1 μM 20E reduced the levels of both mRNA and protein for mitotic cyclin B suggesting an inhibitory role for 20E in cell division. In these cells antimicrobial peptide genes, deptericin and drosomycin were also induced by 20E and the 20E induction was suppressed by JH III both in the presence and absence of peptidoglycan. These data show that Mbn2 cells respond to both 20E and JH and could be an excellent model system to study cross-talk between 20E and JH.