Diazepam is well known for its anxiolytic properties, which are mediated via activation of the GABAA receptor. Diazepam Binding Inhibitor (DBI), also called acyl-CoA binding protein (ACBP), is a ubiquitously expressed protein originally identified based on its ability to displace diazepam from its binding site on the GABAA receptor. Central administration of ACBP or its cleaved fragment, commonly referred to as endozepines, induces proconflict and anxiety-like behaviour in rodents. For this reason, ACBP is known as an anxiogenic peptide. However, the role of endogenous ACBP in anxiety-like behaviour and anxiolytic responses to diazepam has not been investigated. To address this question, we assessed anxiety behaviour and anxiolytic responses to diazepam in two complementary loss-of-function mouse models including astrocyte-specific ACBP KO (ACBP(GFAP) KO) and whole-body KO (ACBP KO) mice. Male and female ACBP(GFAP) KO and ACBP KO mice do not show significant changes in anxiety-like behaviour compared to control littermates during elevated plus maze (EPM) and open field (OF) tests. Surprisingly, ACBP(GFAP) KO and ACBP KO mice were unresponsive to the anxiolytic effect of a low dose of diazepam during EPM tests. In conclusion, our experiments using genetic ACBP loss-of-function models suggest that endozepines deficiency does not affect anxiety-like behaviour in mice and impairs the anxiolytic action of diazepam.