Classical neurotransmitters are mainly known for their roles as neuromodulators, but they also play important roles in the control of developmental and regenerative processes. Here, we used the lamprey model of spinal cord injury to study the effect of serotonin in axon regeneration at the level of individually identifiable descending neurons. Pharmacological and genetic manipulations after a complete spinal cord injury showed that endogenous serotonin inhibits axonal regeneration in identifiable descending neurons through the activation of serotonin 1A receptors and a subsequent decrease in cyclic adenosine monophosphate (cAMP) levels. RNA sequencing revealed that changes in the expression of genes that control axonal guidance could be a key factor determining the serotonin effects during regeneration. This study provides new targets of interest for research in non-regenerating mammalian models of traumatic central nervous system injuries and extends the known roles of serotonin signalling during neuronal regeneration. This article has an associated First Person interview with the first author of the paper.