While recent studies have revealed an involvement of hippocampal interneurons in learning the association among time-separated events, its underlying cellular mechanisms remained not fully clarified. Here, we combined multi-channel recording and optogenetics to elucidate how the hippocampal parvalbumin-expressing interneurons (PV-IN) support associative learning. To address this issue, we trained the mice (box sexes) to learn hippocampus-dependent trace eyeblink conditioning (tEBC) where they associated a light flash conditioned stimulus (CS) with a corneal airpuff unconditioned stimuli (US) separated by a 250-ms time interval. We found that the hippocampal PV-INs exhibited learning-associated sustained activity at the early stage of tEBC acquisition. Moreover, the PV-IN sustained activity was positively correlated with the occurrence of conditioned eyeblink responses at the early learning stage. Suppression of the PV-IN sustained activity impaired the acquisition of tEBC, whereas the PV-IN activity suppre...