Endomorphins (EM-1 and EM-2) are selective, high affinity agonists of the μ-opioid (MOP) receptor, an important target in pain regulation. Their clinical use is impeded by their poor metabolic stability and limited entry to the central nervous system. In this study, the Pro(2) residue of EM-2 was modified systematically through substitution by hydroxyproline (Hyp), (S)-β-homoproline (βPro), 2-aminocyclopentene-1-carboxylic acid (ΔAcpc), or 2-aminocyclohexene-1-carboxylic acid (ΔAchc) to obtain stable MOP active compounds. Both Hyp(2) and βPro(2) substitution decreased receptor affinity. Analogues incorporating alicyclic β-amino acids exhibited diverse receptor binding properties, depending on the configuration of the substituent side-chain. (1S,2R)ΔAcpc(2)-EM-2 was shown to have MOP affinity and selectivity comparable to those of EM-2 and proved to act as agonist while being resistant to proteolysis. NMR and molecular dynamics (MD) studies revealed that bent backbone structures are predominant in the most potent analogues, while their presence is less pronounced in ligands of lower receptor affinity.