Abstract: Fatigue tests were conducted on carbide-free bainitic steel at different stress levels (stress amplitude of 200–550 MPa, stress ratio of −1). The transformation of retained austenite in test steel under alternating loads was studied by scanning electron microscopy, transmission electron microscopy, X-ray diffraction and electron backscatter diffraction. The results show that the critical stress amplitude for transformation of retained austenite in the test steel was 300 MPa. When the stress amplitude was greater than 300 MPa, the retained austenite began to transform into martensite, resulting the decrease in content of austenite and the increase in hardness of the test steel. With the progression of cyclic loading, the content of retained austenite decreased. After fatigue, the Kernel Average Misorientation (KAM) value of retained austenite in the test steel was higher than that of bainitic ferrite, indicating that during fatigue,the retained austenite was more prone to plastic deformation than bainitic ferrite. Compared with the retained austenite between the bainitic ferrite laths with a low Schmid factor, the retained austenite between laths with a high Schmid factor was more likely to transform into martensite during fatigue.