Fmoc-protected Phe-Gly-type (Z)-alkene dipeptide isostere (ADI) and (E)-fluoroalkene dipeptide isostere (FADI) were synthesized and applied to Fmoc-based solid-phase peptide synthesis (SPPS). These cis-peptide bond mimetics were introduced into a bioactive pentapeptide [H-Amb-Phe-Gly-Leu-Arg-Trp-NH(2); Amb = 4-(aminomethyl) benzoic acid], which has potent GPR54 agonistic activity. The resulting pentapeptide derivatives showed low GPR54 agonistic activity, as compared with the parent peptide and (E)-ADI-containing derivative. This suggests that the trans-amide conformer of Phe-Gly peptide bond of the parent peptide would be significantly important for bioactivity. Contrary to our expectations, a (Z)-FADI-containing derivative exhibited essentially no activity, revealing the necessity of critical validation of FADI-bioisosterism.