Aim Histamine receptor H3 has substantial neuropharmacological potential. Currently, knock‐out models of this receptor have been investigated only in mice. We characterized the expression of this receptor in the zebrafish and generated a zebrafish histamine receptor H3 knock‐out line. Using this model, we studied the role of histamine receptor H3 in important behaviours. We also analyzed the effect of histamine receptor H3 knock‐out on monoaminergic systems, which has not been thoroughly studied in any animal model. Methods Generation of a mutant zebrafish line using the CRISPR/Cas9 system. Analysis of locomotor and social behaviour. Expression of histamine receptor H3 was characterized using in situ hybridization. Analysis of monoamine networks using HPLC, immunohistochemistry and quantitative PCR. Results We found that histamine receptor H3 knock‐out zebrafish larvae showed a shorter period of increased locomotion after a sudden onset of darkness, while the knock‐out larvae had a wild type like acute response to sudden darkness. Adult knock‐out fish showed decreased swimming velocity, although locomotor activity of knock‐out larvae was unaltered. Additionally, levels of dopamine and serotonin were significantly decreased in the knock‐out fish, while monoamine‐related gene expression and immunohistochemistry patterns were unchanged. Conclusions Our results show that histamine receptor H3 knock‐out larvae adapt faster to sudden darkness, suggesting a role for this receptor in regulating responses to changes in the environment. The decreased levels of dopamine and serotonin provides the first direct evidence that knock‐out of histamine receptor H3 alters these systems. This article is protected by copyright. All rights reserved.