The physiological function of dopamine is mediated through its G-protein-coupled receptor family. In Drosophila, four dopamine receptors have been molecularly characterized so far. However, due largely to the absence of a suitable preparation, the role of Drosophila dopamine receptors in modulating central synaptic transmission has not been examined. The present study investigated mechanisms by which dopamine modulates excitatory cholinergic synaptic transmission in Drosophila using primary neuronal cultures. Whole-cell recordings demonstrated that cholinergic excitatory postsynaptic currents (EPSCs) were down-regulated by focally applied dopamine (10–500 µm). The vertebrate D1 specific agonists SKF38393 and 6-chloro-APB (10 µm) mimicked dopamine-mediated suppression of cholinergic synaptic transmission with higher potency. In contrast, the D2 agonists quinpirole and bromocriptine did not alter cholinergic EPSCs, demonstrating that dopamine-mediated suppression of cholinergic synaptic transmission is specifically through activation of Drosophila D1-like receptors. Biophysical analysis of miniature EPSCs indicated that cholinergic suppression by activation of D1-like receptors is presynaptic in origin. Dopamine modulation of cholinergic transmission is not mediated through the cAMP/protein kinase A signaling pathway as cholinergic suppression by dopamine occurred in the presence of the protein kinase A inhibitor H-89. In addition, an adenylate cyclase activator, forskolin, led to an increase, not a decrease, of cholinergic EPSC frequency. Finally, we showed that activation of D1-like receptors decreased the frequency of action potentials in cultured Drosophila neurons by inhibiting excitatory cholinergic transmission. All our data demonstrated that activation of D1-like receptors in Drosophila neurons negatively modulates excitatory cholinergic synaptic transmission and thus inhibits neuronal excitability.