The human small heat-shock protein αB-crystallin (αB) rescues misfolded proteins from irreversible aggregation during cellular stress. Binding of Cu(II) was shown to modulate the oligomeric architecture and the chaperone activity of αB. However, the mechanistic basis of this stimulation is so far not understood. We provide here first structural insights into this Cu(II)-mediated modulation of chaperone function using NMR spectroscopy and other biophysical approaches. We show that the α-crystallin domain is the elementary Cu(II)-binding unit specifically coordinating one Cu(II) ion with picomolar binding affinity. Putative Cu(II) ligands are His(83), His(104), His(111), and Asp(109) at the dimer interface. These loop residues are conserved among different metazoans, but also for human αA-crystallin, HSP20, and HSP27. The involvement of Asp(109) has direct implications for dimer stability, because this residue forms a salt bridge with the disease-related Arg(120) of the neighboring monomer. Furthermore, we observe structural reorganization of strands β2-β3 triggered by Cu(II) binding. This N-terminal region is known to mediate both the intermolecular arrangement in αB oligomers and the binding of client proteins. In the presence of Cu(II), the size and the heterogeneity of αB multimers are increased. At the same time, Cu(II) increases the chaperone activity of αB toward the lens-specific protein β(L)-crystallin. We therefore suggest that Cu(II) binding unblocks potential client binding sites and alters quaternary dynamics of both the dimeric building block as well as the higher order assemblies of αB.