While the hippocampus has been implicated in supporting the association among time-separated events,the underlying cellular mechanisms have not been fully clarified.Here,we combined in vivo multi-channel record-ing and optogenetics to investigate the activity of hip-pocampal interneurons in freely-moving mice performing a trace eyeblink conditioning (tEBC) task.We found that the hippocampal interneurons exhibited conditioned stimulus(CS)-evoked sustained activity,which predicted the per-formance of conditioned eyeblink responses (CRs) in the early acquisition of the tEBC.Consistent with this,greater proportions of hippocampal pyramidal cells showed CS-evoked decreased activity in the early acquisition of the tEBC.Moreover,optogenetic suppression of the sustained activity in hippocampal intemeurons severely impaired acquisition of the tEBC.In contrast,suppression of the sustained activity of hippocampal interneurons had no effect on the performance of well-learned CRs.Our findings highlight the role of hippocampal interneurons in the tEBC,and point to a potential cellular mechanism subserving associative learning.