Previously, we have identified a highly potent CXCR4 antagonist 2 [cyclo(-D-Tyr1-Arg2-Arg3-Nal4-Gly5-)] and its Arg2 epimer 3 [cyclo(-D-Tyr1-D-Arg2-Arg3-Nal4-Gly5-)] by the screening of cyclic pentapeptide libraries that were designed based on the structure-activity relationship studies on 14-residue peptidic CXCR4 antagonist 1. In the present study, a new series of analogues of 2 and 3 were synthesized to evaluate the influences of peptide side-chain and backbone modification on bioactivities. Based on the Ala-scanning study, in which each residue in 2 and 3 was replaced with Ala having the identical chirality, substitution of Arg3 and Nal4 [Nal = L-3-(2-naphthyl)alanine] with Ala (compounds 6, 7, 10, 11) led to significant loss of the potency, indicating these amino acids are more important contributors to the bioactivity. For the cyclic peptide backbone, several modifications including d/l-Ala or cyclic amino acids substitution at the Gly5 position and sequential N-methylation on amide nitrogens were conducted. Among the analogues, compounds 13 [cyclo(-D-Tyr1-Arg2-Arg3-Nal4-D-Ala5-)] and 32 [cyclo(-D-Tyr1-D-MeArg2-Arg3-Nal4-Gly5-)] were close in potency to the most potent lead 2. NMR and conformational analysis indicated that both of these analogues favor the same backbone conformation as 2, whereas similar analysis of less potent analogues indicates that an altered backbone conformation is favored. The conformational analysis showed that steric repulsion by a 1,3-allylic strain-like effect across the planar peptide bond might contribute to the conformational preferences of cyclic pentapeptides.