High-cycle fatigue tests were conducted to investigate the effects of temperature, stress ratio (R), specimen orientation, welding and specimen size on the fatigue behavior of type 316L stainless steel. The high-cycle fatigue test results indicated that the fatigue limits significantly decreased when the stress ratio (R) decreased. The corresponding fatigue limits were reduced to lower values when tests were conducted at 300°C, compared to those obtained at room temperature. The fatigue behavior and fatigue limits of standard and subsize specimens were observed to be consistent at both room temperature and 300°C. The constant life diagram was established from the S–N curves acquired. The fatigue limit strongly depended on the materials strength, which was a function of specimen orientation, test temperature, and welding processes. The dimension of the fatigue damaged area on a fracture surface increased as the stress ratio decreased. In the case of R=−1.0, the fatigue damaged region extended over the whole fracture surface. The subgrain boundaries after high-cycle fatigue tests were clearly demonstrated by their diffraction patterns, which were related to the dynamic recovery of multiple dislocations.