In this article, based on analyzing the effect of self-annealing of copper film both empirically and numerically, a new hypothesis has been proposed to better explain the mechanism of copper protrusion evolution with annealing temperature. Electroplated copper in TSV goes through an intense self-annealing process after deposition at room temperature. The effect of self-annealing, which has been all ignored to date, is critical to the annealing behavior of Cu-filled TSV. During self-annealing, a tensile stress field is generated in TSV due to the occurrence of the initial copper protrusion, and this tensile stress can strongly suppress the copper protrusion during annealing treatment. Atomic Force Microscope (AFM) has been used for the measurements of copper protrusion. A finite element model based on the hot deformation and self-annealing of copper film has been proposed to confirm the hypothesis.