AbstractAcyl-CoA binding protein (ACBP) encoded by diazepam binding inhibitor (DBI) is an extracellular inhibitor of autophagy acting on the gamma-aminobutyric acid A receptor (GABA_AR) γ2 subunit (GABA_ARγ2). Here, we show that lipoanabolic diets cause an upregulation of GABA_ARγ2 protein in liver hepatocytes but not in other major organs. ACBP/DBI inhibition by systemically injected antibodies has been demonstrated to mediate anorexigenic and organ-protective, autophagy-dependent effects. Here, we set out to develop a new strategy for developing ACBP/DBI antagonists. For this, we built a molecular model of the interaction of ACBP/DBI with peptides derived from GABA_ARγ2. We then validated the interaction between recombinant and native ACBP/DBI protein and a GABA_ARγ2-derived eicosapeptide (but not its F77I mutant) by pull down experiments or surface plasmon resonance. The GABA_ARγ2-derived eicosapeptide inhibited the metabolic activation of hepatocytes by recombinant ACBP/DBI protein in vitro. Moreover, the GABA_ARγ2-derived eicosapeptide (but not its F77I-mutated control) blocked appetite stimulation by recombinant ACBP/DBI in vivo, induced autophagy in the liver, and protected mice against the hepatotoxin concanavalin A. We conclude that peptidomimetics disrupting the interaction between ACBP/DBI and GABA_ARγ2 might be used as ACBP/DBI antagonists. This strategy might lead to the future development of clinically relevant small molecules of the ACBP/DBI system.