Using high-resolution proton nuclear magnetic resonance (¹H-NMR) and carbon-13 nuclear magnetic resonance (¹³C-NMR) experiments, we have assigned three discernible configurational isomers observed in dimethyl sulfoxide-d₆ (DNSO-d₆) for an antitumor cyclic hexapeptide, RA-VII isolated from Rubia cordifolia. The largest isomer, amounting to 64%, has been assigned as conformer A with only a cis configuration between Tyr-5 and Tyr-6. The second configurational isomer, accounting for 32%, has adopted cis configurations between both Tyr-5 and Tyr-6 and between Ala-2 and Tyr-3. The third isomer, amounting to 4%, was determined to have cis configurations for all of the three N-methyl amide bonds.