Neurotensin (NT) receptor type 2 (NTS2) represents an attractive target for the development of new NT-based analgesics. Here, we report the synthesis and functional in vivo characterization of the first constrained NTS2-selective macrocyclic NT analog. While most chemical optimization studies rely on the NT(8-13) fragment, we focused on NT(7-12) as a scaffold to design NTS2-selective macrocyclic peptides. Replacement of Ile by Leu, and Pro/Pro by allylglycine residues followed by cyclization via ring-closing metathesis led to macrocycle 4, which exhibits good affinity for NTS2 (50 nM), high selectivity over NTS1 (>100 μM), and improved stability compared to NT(8-13). In vivo profiling in rats reveals that macrocycle 4 produces potent analgesia in three distinct rodent pain models, without causing the undesired effects associated with NTS1 activation. We further provide evidence of its non-opioid antinociceptive activity, therefore highlighting the strong therapeutic potential of NTS2-selective analogs for the management of acute and chronic pain.