PF1171 hexapeptides, a family of cyclic hexapeptides produced by fungi, exhibit paralyzing effects on the larvae of silkworms via oral administration. To elucidate the structural features of PF1171 hexapeptides that are crucial for bioactivity, the relationship among 3D structure, bioactivity, and membrane permeability of PF1171F (the peptide with the highest bioavailability) was systematically analyzed through the synthesis of 22 analogues. The PF1171F analogues were prepared by the solid-phase synthesis of a linear precursor and subsequent solution-phase macrolactamization. Analysis by NMR spectroscopy and molecular modeling indicated that the major 3D conformations of PF1171F in various solvents resemble its X-ray crystal structure. The analogues with this conformation tend to exhibit potent paralysis against silkworms, indicating the significance of the conformation in the paralysis. The biological activity was dependent on the mode of administration, varying between hemolymph injection and oral administration. Parallel artificial membrane permeability assay (PAMPA) of the analogues revealed a correlation between membrane permeabilities and paralytic activity by hemolymph injection, indicating that the target molecule of PF1171F is present inside the cell membrane.