In our earlier paper (Ghosh et al., 2013), we have shown that (i) the positively charged hen egg whitelysozyme (HEWL), dispersed in water, binds electrostatically with the negatively functionalized ironoxide nanoparticles (IONPs), and (ii) the Na⁺counterions, associated with functionalized IONPs, dif-fuse into bound proteins and irreversibly unfold them. Having this information, we have extended ourinvestigation and report here the effect of the size and the charge of alkaline metal counterions onthe conformational modification of HEWL. In order to obtain a negative functional ‘shell’ on IONPs andthe counterions of different size and charge we have functionalized IONPs with different derivatives ofcitrate, namely, tri-lithium citrate (TLC, Li₃C₆H₅O₇), tri-sodium citrate (TSC, Na₃C₆H₅O₇), tri potassiumcitrate (TKC, K3C6H5O7) and tri-magnesium citrate (TMC, Mg₃C₁₂H₁₀O₁₄). The size of counterions variesas Mg²⁺< Li⁺< Na⁺< K⁺. After interaction with the functionalized IONPs, the unfolding of HEWL was themaximum in presence of Li⁺, and was decreasing with increasing size of counterions. The UV–vis absorp-tion measurements indicated that the unfolding of HEWL was due to modification in the hydrophobicenvironment around the tryptophan regions. The unfolding of HEWL was associated with the change offolding conformation from the -helix to the -sheet. In absence of counterions, ligand-IONPs have noeffect on the native conformation of HEWL. An effective use of counterions in order to modify proteinconformation (and, the functionality) via protein–nanoparticle electrostatic interaction is a new finding,and be useful for an alternative medical therapy.