The hippocampus is a spatial learning and memory hub underpinned by long-term potentiation (LTP). However, it remains unclear how neuropeptides shape the process. Dual-optogenetic stimulation enables us to differentiate the roles of different projections in LTP induction. Ex vivo recordings showed theta-burst stimulation (TBS) of neither Schaffer collateral (SC) nor medial entorhinal cholecystokinin (MECCCK) projection alone optogenetically could induce homosynaptic LTP in their own pathway. CA3-CA1 LTP could only be induced hetero-synaptically when SC activation is paired with TBS of the MECCCK projections, which released CCK. MECCCK neurons showed higher activity and likely released CCK during exploration. Systematic knockout of CCK impaired LTP and spatial-related task performance. Selective inhibition of the MECCCK neurons or down-regulation of their Cck mRNA level impaired LTP formation and behavioural performance. Our results reveal an enabling role of MEC CCKergic modulation in TBS-induced CA3-CA1 LTP and spatial memory.