In order to clarify the effects of a great amount of static pre-strain (1/4 cycle) on fatigue life of AISI 316L stainless steel, strain controlled low cycle fatigue tests were carried out. Hardness and fracture ductility of specimens were measured. Results showed that in the case of a specimen with Δεp=0.01, static pre-strain of 0.16 noticeably reduced a fatigue life. To clarify the reasons for the decrease in fatigue life with pre-strain, the maximum stress amplitude of the hysteresis loop was measured. In the case of the specimen with Δεp=0.01, work softening was observed in the early stage of fatigue life. On the other hand, when the cyclic plastic strain ranges were Δεp=0.10, no softening was observed. These results suggest that work softening is significant in determining the fatigue life of the specimens at a relatively low level of applied cyclic strain.