Long-lasting forms of synaptic plasticity involve modification of presynaptic strength in many brain regions. Although a presynaptic site for expression is well established, the detailed molecular mechanisms that lead to sustained changes in neurotransmitter release remain unclear. Here, we use acute in vivo genetic manipulation of synaptic proteins to investigate the molecular basis for presynaptic long-term potentiation (LTP) at hippocampal mossy fiber synapses. Munc13 proteins are active zone proteins that are essential for synaptic vesicle priming and neurotransmitter release. Munc13 proteins also interact with RIM1alpha, an active zone protein required for presynaptic long-term plasticity. By taking advantage of the observation that the RIM-binding domain of Munc13 is separable from the domain that is required for neurotransmitter release, we selectively tested whether Munc13-1 is an effector for RIM1alpha in presynaptic LTP. Our results provide the first evidence for the involvement of Munc13-1 in presynaptic long-term synaptic plasticity. We further demonstrate that the interaction between RIM1alpha and Munc13-1 is required for this plasticity. These results advance our understanding of the molecular mechanisms of presynaptic plasticity and suggest that modulation of vesicle priming may provide the cellular substrate for expression of LTP at mossy fiber synapses.