Recently, mixed opioid/NOP agonists came to the spotlight for their favorable functional profiles and promising outcomes in clinical trials as novel analgesics. This study reports on two novel chimeric peptides incorporating the fragment Tyr-c[D-Lys-Phe-Phe]Asp-NH (), a cyclic peptide with high affinity for µ and κ opioid receptors (or MOP and KOP, respectively), conjugated with the peptide Ac-RYYRIK-NH, a known ligand of the nociceptin/orphanin FQ receptor (NOP), yielding RP-170-RYYRIK-NH () and RP-170-Gly-RYYRIK-NH (). In vitro, the chimeric gained affinity for KOP, hence becoming a dual KOP/MOP agonist, while behaved as a mixed MOP/NOP agonist with low nM affinity. Hence, was selected for further in vivo experiments. Intrathecal administration of this peptide in mice elicited antinociceptive effects in the hot-plate test; this action was sensitive to both the universal opioid receptor antagonist naloxone and the selective NOP antagonist SB-612111. The rotarod test revealed that administration did not alter the mice motor coordination performance. Computational studies have been conducted on the two chimeras to investigate the structural determinants at the basis of the experimental activities, including any role of the Gly spacer.