Plastic deformation and hardening mechanism of nuclear grade austenitic stainless steel, SUS316NG, at room and cryogenic temperatures were investigated using advanced indentation method and observations by transmission electron microscope (TEM). First, tensile and compressive pre-strains were applied at room temperature up to true strain values of 20%. Detailed indentation testing revealed that the strain hardening was detected when the indentation sizes were more than 50 μm, while it disappeared when the indentation size decreased down to 1 μm. This behavior was explained by the slip band size due to dislocation structure. Dual indenter method successfully predicted the stress-strain response of the pre-strained specimens. Second, Charpy impact tests were performed at room temperature and 77 K. The fracture toughness was sufficiently large at 77 K, however, ductility was slightly decreased compared with that at room temperature. Martensitic transformation was observed near the notch tip for the specimen tested at 77 K. This hardening was detectable by the indentation testing even if the indentation size was less than 1 μm.