Reward processing is an essential function for survival of individuals. Aberrant reward processing induces an objectively incorrect judgment about the value of reward, which ultimately leads to drug addiction and anhedonia. Median raphe nucleus (MRN), a serotonergic nucleus, is suggested to play a critical role in reward processing. However, it is not fully understood whether MRN serotonin neurons respond to reward and how they regulate reward-related behavior. Here, we determined the response of MRN serotonin neurons to reward and aversive stimuli and the causal link between their activity and behavioral changes by using optogenetic recordings and manipulation. GCaMP6s, a fluorescent Ca²⁺ indicator, was expressed in MRN serotonin neurons to monitor neuronal activity. We measured the fluorescence in MRN by fiber photometry before and after sucrose intake as reward stimuli and tail pinch as aversive stimuli. The fluorescence decreased after sucrose intake, while increased after pinch. We also found the fluorescence of MRN axons in interpeduncular nucleus (IPN), a major projection area of MRN, decreased after sucrose intake. Moreover, optogenetic inactivation of MRN serotonin neurons increased time spent in the stimulation-paired chamber in conditioned place preference test, while their activation shortened spent time in the stimulation-paired chamber. Optogenetic intervention of MRN-IPN pathway had an effect similar to soma manipulation. Our results suggest the MRN serotonin neurons projecting to the IPN play a key role in the reward processing.