The dopaminergic system plays critical roles in Drosophila olfactory associative learning. In this study, we identified DAN-c1, a single dopaminergic neuron (DAN) in each brain hemisphere, that is both necessary and sufficient for Drosophila larval aversive associative learning. Compared to well-known roles of excitatory D1-like receptors in learning, the role of D2-like receptors (D2Rs) has not been fully investigated. We observed that D2Rs were expressed in DANs and the mushroom body (MB) in third instar larval brains. Knockdown of D2Rs in DAN-c1 by microRNA impaired aversive learning. Optogenetic activation of DAN-c1 during training led to an aversive learning deficit as well, indicating that D2R achieves its functions via autoreceptor inhibition. Interestingly, knockdown of D2R in MB impaired both appetitive and aversive learning. These results reveal that D2Rs in different brain structures play important but distinct roles in Drosophila larval olfactory learning, providing new insights into molecular mechanisms underlying associative learning.