α-Casozepine (α-CZP) is an anxiolytic-like bioactive decapeptide derived from bovine αs1‑casein. The N-terminal peptide YLGYL was previously identified after proteolysis of the original peptide in an in vitro digestion model. Its putative anxiolytic-like properties were evaluated in a Swiss mice model using a light/dark box (LDB) after an intraperitoneal injection (0.5 mg/kg). The effect of YLGYL on c-Fos expression in brain regions linked to anxiety regulation was afterwards evaluated via immunofluorescence and compared to those of α-CZP and diazepam, a reference anxiolytic benzodiazepine. YLGYL elicited some anxiolytic-like properties in the LDB, similar to α‑CZP and diazepam. The two peptides displayed some strong differences compared with diazepam in terms of c-Fos expression modulation in the prefontal cortex, the amygdala, the nucleus of the tractus solitarius, the periaqueductal grey, and the raphe magnus nucleus, implying a potentially different mode of action. Additionally, YLGYL modulated c-Fos expression in the amygdala and in one of the raphe nuclei, displaying a somewhat similar pattern of activation as α-‑CZP. Nevertheless, some differences were also spotted between the two peptides, making it possible to formulate the hypothesis that these peptides could act differently on anxiety regulation. Taken together, these results showed that YLGYL could contribute to the in vivo overall action of α‑CZP.