Cytochrome c, an iron containing metalloprotein in the mitochondria of the cells with an oxide/redox property, plays key role in the cell apoptotic pathway. In this study, the interaction of silver nanoparticles (AgNPs) with cytochrome c (Cyt c) was investigated by using a combination of spectroscopic, imaging and thermodynamic techniques, including dynamic light scattering (DLS), ultraviolet-visible (UV-vis) spectroscopy, transmission electron microscopy (TEM), fluorescence spectroscopy, near and far circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC). DLS and UV-vis analysis evidenced the formation of surface complexes of Cyt c on AgNPs. The saturation of surface coverage of AgNPs was observed at 4.36 Cyt c molecules per nm 2 of AgNPs. The surface complexation resulted in a promotion of the Ag dissolution overtime. The negative sign of enthalpic (ΔH) contribution suggested that electrostatic forces are indicative forces in the interaction between protein and AgNPs. Moreover, the fluorescence spectra revealed that the conformation of protein was altered around tryptophan (Trp) and tyrosine (Tyr) residues indicating the alteration of the tertiary structure of Cyt c. CD analysis evidenced that the secondary structure of Cyt c was modified under AgNPs-Cyt c interactions and the binding of Cyt c onto AgNPs resulted in remarkable structural perturbation around the active site heme, which in turn alter the protein enzymatic activity. The results of the present study contributed to a deeper insight on the mechanisms of interaction between NPs and biomacromolecules and could help establish the in vivo fate of AgNPs on cellular redox homeostasis.