Accumulating evidence indicates that the laterodorsal tegmental nucleus (LDT) is associated with reward processing and addiction. The cholinergic projection from the LDT to the ventral tegmental area is essential for a large dopamine release in the nucleus accumbens, which is critically involved in the reinforcing effects of addictive drugs, including cocaine. In contrast to the large number of studies on plasticity induced after cocaine exposure in the mesocorticolimbic dopaminergic system, it remains unknown whether LDT cholinergic neurons exhibit plastic changes following cocaine administration. To address this issue, we performed ex vivo whole-cell recordings in LDT cholinergic neurons obtained from rats following cocaine administration. Neurons obtained from 1 day after 5-day cocaine-treated rats showed significantly smaller paired-pulse ratios of evoked EPSCs and higher miniature EPSC frequencies than those from saline-treated rats, indicating an induction of presynaptic plasticity of increased glutamate release. This plasticity seemed to recover after a 5-day withdrawal from repeated cocaine exposure, and required NMDA receptor stimulation and nitric oxide production. Additionally, pharmacological suppression of activity of the medial prefrontal cortex inhibited the presynaptic plasticity in the LDT. On the other hand, AMPA/NMDA ratios were not different between saline- and cocaine-treated groups, revealing an absence of postsynaptic plasticity. These findings provide the first direct evidence of cocaine-induced synaptic plasticity in LDT cholinergic neurons and suggest that the presynaptic plasticity enhances the activity of LDT cholinergic neurons, contributing to the expression of cocaine-induced addictive behaviors through the dysregulation of the mesocorticolimbic system.