The effects of prestraining on high-cycle fatigue strength of newly developed low alloy TRIP-aided steels with different matrix structures and different retained austenite characteristics were investigated for automotive applications. Prestraining to 10% in tension increased the fatigue limit of the TRIP-aided steels, especially in steel with a polygonal ferrite matrix. It was considered that the polygonal ferrite matrix brought on high a fatigue limit mainly due to TRIP of the retained austenite and high compressive internal stress in the matrix resulting from a hard second phase on prestraining. On the other hand, the steel with bainitic ferrite lath matrix exhibited only a small increase in the fatigue limit after prestraining. This was expected to be mainly due to “strain-induced martensite hardening” on prestraining, with small contributions of TRIP of compressive internal stress in the matrix. In addition, a very interesting finding was obtained that the internal stress is the most effective parameter among some parameters to increase the fatigue strength in low alloy TRIP-aided steels.