The abundantly expressed neuropeptide Y (NPY) plays an important role in anxiety and stress reactivity, as exogenous NPY administration reduces anxiety-like behaviour in rodents. However, unlike the potent effects of NPY seen in pharmacological studies, two independent examinations of a genetic mouse model for NPY deficiency have shown only subtle, inconsistent and task-dependent anxiety-related phenotypes for male mutants. Here we present results of a newly developed germline NPY-knockout model, which has been characterized behaviourally using a comprehensive multi-tiered phenotyping strategy. Mice of both sexes were investigated in locomotion and exploration tasks, anxiety-related paradigms, a hippocampus-dependent memory test and a battery of basic tasks screening for sensory and motor functions. Male and female NPY-deficient mice consistently demonstrated suppressed levels of locomotion and exploration. Furthermore, mutant mice exhibited a pronounced anxiogenic-like phenotype when tested in spatiotemporal anxiety-relevant paradigms (i.e. elevated-plus maze, open field and light–dark task). Importantly, this phenotype was more pronounced in male NPY mutants, revealing a moderate sexually dimorphic impact of NPY deficiency on behaviour. Interestingly, lack of NPY did not result in impaired learning and memory in either sex. Our carefully selected comprehensive behavioural phenotyping strategy revealed a consistent hypolocomotive and sex-dependent anxious-like phenotype. This new NPY-knockout mouse model reveals the importance of sex-specific testing. It also offers a potent new model for research into anxiety-related disorders and suggests potential treatment options for these conditions via the NPY system.