Histamine plays an important role in the control of brain functions. Our recent study showed that histamine N-methyltransferase (HNMT), a histamine-metabolizing enzyme, regulates brain histamine concentration. Although previous study indicated the expression of HNMT in neurons, the contribution of neuronal HNMT to brain histamine system is still unknown. In the present study, we phenotyped neuron-specific Hnmt knockout (cKO) mice to clarify the importance of neuronal Hnmt. First, we generated cKO mice by injecting AAV9-hSyn1-Cre, which express Cre recombinase specifically in neurons, to Hnmt flox mice. Histamine concentration of cKO brains was significantly elevated compared to that of control brains, indicating the importance of neuronal Hnmt for brain histamine concentration. Behavioral test battery demonstrated that cKO mice showed reduced anxiety-like behaviors, reduced depression-like behaviors and increased locomotor activity in novel and familiar environment. These data show the importance of neuronal Hnmt for brain histamine system and brain functions.