Ultrasonic vibrations with ultrasonic powers of 0 W, 600 W, 1000 W and 1400 W were applied during laser melting deposition of 316L stainless steel. The effects of ultrasonic powers on the grain morphologies and sizes, the microstructure formation mechanism and grain growth characteristics of 316L stainless steel were studied. The results show that the application of ultrasonic vibrations can break the directional epitaxial growth of large sized columnar grains, resulting in a refinement of microstructure, and increases of cumulative misorientation and dislocation densitie along the long axis of grains. By promoting the convections in the melt pool, and thus reducing the temperature gradient along the depostion direction, the application of ultrasonic vibrations accelerate the transitions of growth directions for columnar grains. The application of ultrasonic vibrations also help to increase the cooling rate during solidification, resulting in refinements of both columnar grains and the interior columnar dendrites.