The neuromodulators dopamine (DA) and serotonin (5-hydroxytryptamine; 5HT) powerfully regulate associative learning1–8. Similarities in the activity and connectivity of these neuromodulatory systems have inspired competing models of how DA and 5HT interact to drive the formation of new associations9–14. However, these hypotheses have not been tested directly because it has not been possible to interrogate and manipulate multiple neuromodulatory systems in a single subject. Here, we establish a mouse model enabling simultaneous genetic access to the brain’s DA and 5HT neurons. Anterograde tracing revealed the nucleus accumbens (NAc) to be a putative hotspot for the integration of convergent DA and 5HT signals. Simultaneous recording of DA and 5HT axon activity, together with genetically encoded DA and 5HT sensor recordings, revealed that rewards increase DA signaling and decrease 5HT signaling in the NAc. Optogenetically dampening DA or 5HT reward responses individually produced modest behavioral deficits in an appetitive conditioning task, while blunting both signals together profoundly disrupted learning and reinforcement. Optogenetically reproducing DA and 5HT reward responses together was sufficient to drive acquisition of new associations and supported reinforcement more potently than either manipulation alone. Together, these results demonstrate that striatal DA and 5HT signals shape learning by exerting opponent control of reinforcement.