Pressure strengthening (PS) technology, also called cold stretching, is widely used to increase the allowable stress and reduce the weight of cryogenic pressure vessels (CPVs) made of austenitic stainless steels (ASSs). However, with taking accounts of the manufacturing process and service environment of CPVs, the main source of the strengthening effect for yielding strength improvement is still unclarified. Therefore, in this work, the effect of the 9% prestrain and low-temperature (−196 °C) on cryogenic mechanical properties of S30408 stainless steel was comparatively investigated by cryogenic tensile test and martensitic transformation measurement. After evaluating the main source of strengthening increments, the effect of prestraining on welded joints was studied by Vickers microhardness test. Then the engineering significance of performing the PS on a cryogenic inner vessel was explored based on finite element analysis (FEA). It revealed that at −196 °C, the strengthening effect of low-temperature plays a leading role for strength increments, though the yield strength can be increased obviously by prestraining at room temperatures. Meanwhile, the performing of 9% prestrain could enhance the distribution uniformity of the Vickers microhardness of welded joints. Furthermore, the prior operation of PS on inner vessels is beneficial to prevent further plastic deformation during the subsequent hydraulic test, avoid thermal insulation degradation of the vacuum jackets and even stress concentrations in roots of pipes, which is also of engineering significance to smooth the subsequent processes such as the assembly with outer containers and other product tests.