A macrocyclic peptide scaffold with well-established structure-property relationship is desirable for tackling undruggable targets. Here, we adopted a natural macrocycle, cyclosporin O (<b>CsO</b>) and its derivatives (<b>CP1</b>-<b>3</b>), and evaluated the impact of conformation on membrane permeability, cyclophilin A (CypA) binding, and the pharmacokinetic (PK) profile. In nonpolar media, <b>CsO</b> showed a similar conformation to cyclosporin A (<b>CsA</b>), a well-known chameleonic macrocycle, but less chameleonic behavior in a polar environment. The weak chameleonicity of <b>CsO</b> resulted in decreased membrane permeability; however, the more rigid conformation of <b>CsO</b> was not detrimental to its PK profile. <b>CsO</b> exhibited a higher plasma concentration than <b>CsA</b>, which resulted from minimal CypA binding and lower accumulation in red blood cells and moderate oral bioavailability (<i>F</i> = 12%). Our study aids understanding of <b>CsO</b>, a macrocyclic peptide that is less explored than <b>CsA</b> but with greater potential for diversity generation and rational design.