Accumulating evidence shows that dopaminergic neurons of the ventral tegmental area (VTA) are crucial for rewarding and aversive responses. The activity of these neurons is intricately regulated by various neuropeptides, one of which is the endogenous opioid dynorphin. It is thought that the main source of dynorphin in the VTA arises from nucleus accumbens (NAc) D1-MSNs, and that dynorphin acts primarily on kappa opioid receptors (KORs), serving as negative feedback mechanism to modulate dopamine release. Thus, dynorphin signals may regulate dopamine dynamics and, in this way, contribute for rewarding and aversive learning. However, due to the challenge of measuring endogenous opioids in vivo with sufficient temporal and spatial resolution, there are no studies focusing on dynorphin dynamics in the VTA during associative learning. Here, we used a newly developed fluorescent dynorphin sensor, kLight1.3, to characterize VTA dynorphin signals during associative learning. We were able to trigger dynorphin release in the VTA by optogenetic activation of NAc neurons, confirming NAc-specific release. In addition, we found dynamic release of dynorphin across days of learning of both appetitive and aversive Pavlovian conditioning task. When applying different strategies to downregulate dynorphin action in the VTA, we observed that disrupting dynorphin signals in the VTA significantly impairs the acquisition of appetitive and aversive learning behaviors.Together, these findings underscore the pivotal role of VTA dynorphin/KOR signaling in shaping rewarding and aversive learning.