Nanoindentation measurements are made on an interstitial-free steel to reveal the effect of single grain boundary on deformation behavior. Three different sites, “on grain boundary”, “near grain boundary” and “grain interior”, were probed to investigate the pop-in behavior on the initial loading curve and nanohardness. The typical pop-in load at the “grain interior” gives a maximum shear stress beneath the indenter as an order of ideal strength. The pop-in load at the “on grain boundary” is significantly smaller than that at the other sites, indicating that the grain boundary acts as an effective dislocation source with a lower applied shear stress. The nanohardness in the “grain interior” is about 20% lower than that at the other sites, suggesting that a single grain boundary has significant resistance to indentation-induced deformation.