Protein orientation in nanoparticle-protein conjugates plays a crucial role in binding to cell receptors and ultimately, defines their targeting efficiency. Therefore, understanding fundamental aspects of the role of protein orientation upon adsorption on the surface of nanoparticles (NPs) is vital for the development of clinically important protein-based nanomedicine. In this work, new insights on the effect of the different orientation of cytochrome c (cyt c) bound to gold nanoparticles (GNPs) using various ligands on its apoptotic activity is reported. Time-of-Flight Secondary-Ion Mass Spectrometry (ToF- SIMS), electrochemical and circular dichroism (CD) analyses are used to investigate the characteristics of cyt c orientation and structure on functionalized GNPs. These studies indicate that the orientation and position of the heme ring inside the cyt c structure can be altered by changing the surface chemistry on the NPs. A difference in the apoptosis inducing capability because of different orientation of cyt c bound to the GNPs is observed. These findings indicate that the biological activity of a protein can be modulated on the surface of NPs by varying its adsorption orientation. This study will impact on the rational design of new nanoscale biosensors, bioelectronics, and nanoparticle-protein based drugs.