Hilar mossy cells (MCs) of the dentate gyrus receive inputs from dentate granule cells (DGCs) and project their glutamatergic axons back to ipsilateral and contralateral DGCs along the longitudinal axis of the hippocampus, thereby establishing a positive feedback loop. In addition, MCs make synapses with hilar GABAergic interneurons, generating feed-forward inhibition onto DGCs. While MCs have been implicated in epileptogenesis and could participate in some forms of memory, little is known about dynamic changes in synaptic strength at monosynaptic and disynaptic pathways from MC to DGCs. Here, we investigated whether MC-DGC synapses can undergo long-term, activity-dependent forms of plasticity. To this end, we performed whole-cell patch clamp recordings from DGCs in acute hippocampal slices of young-adult rats. EPSCs were monitored in the presence of the GABA_A receptor antagonist picrotoxin while presynaptic MC fibers (MCFs) were activated by local extracellular stimulation in the inner third of the dentate molecular layer. We found that repetitive stimulation of MCFs using multiple 50 Hz-bursts induced long-term potentiation (LTP) of MCF-EPSCs (MCF-LTP). We found that presynaptic cAMP/PKA signaling and postsynaptic Ca²⁺ elevation, but not NMDA receptor activation are required for MCF-LTP. We next used optogenetic methods to selectively activate MCFs. We injected AAV carrying channelrhodopsin-2 (ChR2) into the ipsilateral hilus and examined contralateral hippocampal slices. Activation of ChR2-expressing commissural MCF terminals by light illumination elicited biphasic EPSC-IPSC responses under normal recording conditions. We confirmed that IPSCs are disynaptic responses via hilar interneurons. By delivering high frequency light illumination, LTP was selectively induced at EPSCs but not at disynaptic IPSCs. Finally, we tested the effects of MCF-LTP on DGC output. In these experiments, DGC firing was monitored by brief burst activation of MCFs by electrical stimulation. We found that DGC firing was enhanced after induction of MCF-LTP. Taken together, our findings indicate that MCF-LTP changes excitation/inhibition balance and modulates output of DGCs.