Ligand-protected metallic clusters exhibit optical activity when chiral molecules are used as protecting units. Various mechanisms, such as the inherently chiral metallic cluster core, the dissymmetric field effect, and the chiral footprint model, have been proposed as possible explanations of the nonzero circular dichroism (CD) spectra found for these nanoscale materials. This communication presents a first-principles theoretical study of the CD spectrum of the Au25(SR)18− cluster that was undertaken to gain insight into the physicochemical origin of the optical activity measured for the glutathione-protected Au25(SG)18− cluster. The calculated CD spectrum of the cysteine-protected cluster, with Rcys = CβH2−CαH(NH2)−COOH, shows good agreement with the experimental data obtained for the glutathione-protected cluster. Analysis of the calculated CD spectra of the peculiar two-shell metallic core and the two distinct thiolate−Au binding modes existing in the Au25(SRcys)18− cluster showed that the weak CD signal due to the slight distortion of cluster core is enhanced by the dissymmetric location of the ligands forming the Au−S binding modes. This result shows that the mechanisms proposed to explain the optical activity of chiral-ligand-protected metallic clusters cannot be differentiated but are acting concurrently. It is also predicted that the CD line shape should be highly sensitive to the orientation of the thiolate ligands forming the cluster protecting layer and to the stability of the thiolate−Au binding modes.