The nucleus accumbens (NAc) is a key brain region involved in rewarding and aversive responses. The NAc is mainly composed of GABAergic medium spiny neurons (MSNs) expressing either dopamine receptor D1 or D2. Some studies propose an antagonist role of D1- and D2-MSNs, whereas others suggest that both populations play distinct, but not necessarily opposing roles in valence behaviour.Here, we used deep brain 1-photon calcium imaging to follow NAc D1- or D2-MSN activity in response to opposing valence stimuli and during associative learning. In addition, we used optogenetics to further unravel the contribution of each population for associative learning. We show that despite stochastic encoding at individual neuronal level, NAc D1- or D2-population activity encodes positive and negative valence unconditioned stimuli. Supporting a model of co-activation, we found that the two populations form similar functional clusters during Pavlovian conditioning. Moreover, we found that D2-MSNs signal reward omission and extinction more prominently than D1-MSNs, supporting a key role of these neurons in monitoring and updating outcome information. In line with this hypothesis, optogenetic inhibition of D2-MSNs delays extinction of aversive Pavlovian associations.In sum, we showed that population activity of either MSNs can be used to represent and discriminate positive and negative valence stimuli. Our data strongly favours a model which the two subpopulations are co-recruited to encode CS-US associations, and drive motivated behaviors.