Synaptic potentiation in Drosophila is observed at cholinergic synapses between antennal lobe (AL) and mushroom body (MB) neurons in the adult brain; however, depression at the AL-MB synapses has not yet been identified. By ex vivo Ca2+ imaging in an isolated cultured Drosophila brain, we found novel activity-dependent depression at the AL-MB synapses. The degree of Ca2+ responses after repetitive AL stimulation is significantly reduced in the dendritic region of MB neurons (calyx) compared with those before AL stimulation, and this reduction of Ca2+ responses remains for at least 30 min. The expression of rutabaga, which encodes Ca2+ /calmodulin-dependent adenylyl cyclase, is essential in the MB neurons for the reduction of Ca2+ responses in the calyx. Our study reveals that elevation of cAMP production in the calyx during repetitive AL stimulation induces the depression at the AL-MB synapses.