It is crucial for animals to select an appropriate behavior in a conflict situation where they face both positive and negative motivation. But the neural circuit mechanism underlying behavioral selection in a conflict situation is unclear. We set up a behavioral paradigm in which mice need to explore an experimental context where they might receive electric shocks in order to obtain sucrose solution. Using c-Fos expression as a marker of neuronal activation, we found that the anterior cingulate cortex (ACC) and periaqueductal gray (PAG) were activated in the conflict situation. Anterograde and retrograde tracing with AAV-CaMKII-eYFP and Fluoro-Gold, respectively, revealed dense projections from the ACC to the PAG. To record ACC-PAG activities from behaving mice, a genetically encoded calcium indicator, GCaMP6, was expressed selectively in the ACC-PAG pathway and its fluorescence was detected through an optic fiber. ACC-PAG activity was associated with sucrose-seeking behavior. Furthermore, optogenetic activation of the ACC-PAG pathway shortened the latency to obtain sucrose. These results suggest that ACC inputs to the PAG facilitate reward-seeking behavior in the conflict context.