Nitriding of an austenitic stainless steel (ASS) at moderate temperature (400 °C) increases significantly its hardness without loss of corrosion resistance. This study presents the first comparison between single crystalline (,  and  orientations) and polycrystalline 316L ASS ion beam nitriding. The current density, ion energy and temperature were 0.5 mA·cm−2, 1.2 keV and 400 °C, respectively. The nitrogen distribution profiles were measured by nuclear reaction analysis (NRA). A notable difference of nitrogen penetration between polycrystalline and single-crystalline samples is observed, depending on the orientation of the single crystals. The nitrogen penetration is higher for  and  oriented single crystals. X-ray diffraction (XRD) reveals the presence of the γN phase in both types of samples. Moreover, it shows that the nitrided layer in single crystals exhibits disorientations in relation to the matrix.