The effect of ultrasonic treatment on the microstructural evolution and the related softening process in tensile pre-deformed 316 stainless steel was studied by means of electron backscatter diffraction method, optical microscopy, and microhardness measurement. It was observed that different levels of ultrasonic energy induced complex microstructural changes in the treated samples. A large decrease in twin boundaries was observed, which is an indication of the de-twinning process under ultrasonic treatment. A new mechanism for the de-twinning process under oscillatory stress of ultrasonic vibration was proposed. It was shown that de-twinning under ultrasonic treatment led to dislocation production from twin boundaries. Inverse pole figures investigation revealed strong grain rotation following ultrasonic treatment in tensile pre-deformed samples. Subgrain formation in the ultrasonic treated austenitic stainless steel samples indicated that considerable ultrasonic energy was induced by the ultrasonic vibration, which provided the activation energy needed for dislocation climb and cross-slip. The ultrasonic induced subgrain formation, dislocation annihilation, and de-twinning, which resulted in a decrease of the microhardness in the samples, can be considered as possible mechanisms for the acoustic softening in the austenitic stainless steels.