Food protein-derived peptides with agonistic effects on receptors have great potential for treating anxiety, hypertension, and stress. In the present study, ****** peptides with agonistic activities for δ-receptor-expressing HEK293 cells were screened from casein hydrolysates prepared with five types of food grade proteolytic enzymes, among which casein hydrolysate with Aspergillus oryzae protease ASD showed the highest ****** activity. Eluted fractions showing potent ****** activity were further purified for active peptides by reverse phase-HPLC. The peptide in the active fraction was identified as YPFPGPIPNS, a member of β-casomorphin (CM-10) (β-casein 60–69). Various CM-10 derivative peptides were synthesized and their characteristic features for specificities towards δ- and μ-receptors were determined. Peptides 5 to 12 amino acids long showed relatively higher ****** activities for δ- and μ-receptors. CM-10 was docked into the optimized δ-receptor model. The CDOCKER energies of the CM-10 derivatives were consistent with their ****** activities. In the elevated plus-maze study, CM-10 showed a significant anti-anxiety effect in BALB/c mice at a dose of 10 mg per kg body weight when administered orally, but not via intravenous injection. Furthermore, intravital imaging revealed that Ca²⁺ signaling was induced in the small intestinal villi of a Yellow Cameleon 3.60 (YC3.60)-expressing mouse upon injection with CM-10. However, this decreased in the presence of δ- or μ-receptor antagonists. These results suggest that the ****** peptide CM-10 prepared from casein with ASD has an anti-anxiety effect through interaction with gut δ- and/or μ-****** receptors in the mouse gut.