Abstract Sensory inputs are subjected to modulation by central neural networks involved in controlling movements. It has been shown that serotonin (5‐HT) modulates sensory transmission. This study examines in lampreys the effects of 5‐HT on sensory transmission to brainstem reticulospinal (RS) neurons and the distribution of 5‐HT cells that innervate RS cells. Cells were recorded intracellularly in the in vitro isolated brainstem of larval lampreys. Trigeminal nerve stimulation elicited disynaptic excitatory responses in RS neurons, and bath application of 5‐HT reduced the response amplitude with maximum effect at 10 μ m . Local ejection of 5‐HT either onto the RS cells or onto the relay cells decreased sensory‐evoked excitatory postsynaptic potentials (EPSPs) in RS cells. The monosynaptic EPSPs elicited from stimulation of the relay cells were also reduced by 5‐HT. The reduction was maintained after blocking either N ‐methyl‐ d ‐aspartate (NMDA) or α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) receptors. The local ejection of glutamate over RS cells elicited excitatory responses that were only slightly depressed by 5‐HT. In addition, 5‐HT increased the threshold for eliciting sustained depolarizations in response to trigeminal nerve stimulation but did not prevent them. Combined 5‐HT immunofluorescence with axonal tracing revealed that the 5‐HT innervation of RS neurons of the middle rhombencephalic reticular nucleus comes mainly from neurons in the isthmic region, but also from neurons located in the pretectum and caudal rhombencephalon. Our results indicate that 5‐HT modulates sensory transmission to lamprey brainstem RS cells.