Histamine H3 receptors (H3Rs) were first characterized as autoreceptors modulating histamine release and synthesis via negative feedback. Acute H3R stimulation or blockade with selective agonists and antagonists suggests a role for H3R in anxiety and cognition. However, little is known about the long-term effects of H3R blockade on brain function. In the current study, mice lacking H3 receptors (H3R−/−) were used to investigate the role of H3R-mediated signalling in anxiety and cognition. H3R−/− mice showed enhanced spatial learning and memory in the Barnes maze. In addition, H3R−/− mice showed reduced measures of anxiety in the elevated plus and zero mazes involving exploratory behaviour and avoidable anxiety-provoking stimuli, but enhanced acoustic startle responses involving unavoidable anxiety-provoking stimuli. These behavioural alterations were associated with higher arginine vasopressin levels in the central and basolateral nuclei of the amygdala. These findings support a role for H3Rs in mediating histamine effects on spatial learning and memory and measures of anxiety.