Diazepam is well known for its anxiolytic properties, which are mediated via activation of the GABA_A 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 GABA_A 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.