Mitotic phosphorylation of the spindle checkpoint component BubR1 is highly conserved throughout evolution. Here, we demonstrate that BubR1 is phosphorylated on the Cdk1 site T620, which triggers the recruitment of Plk1 and phosphorylation of BubR1 by Plk1 both in vitro and in vivo. Phosphorylation does not appear to be required for spindle checkpoint function but instead is important for the stability of kinetochore-microtubule (KT-MT) interactions, timely mitotic progression, and chromosome alignment onto the metaphase plate. By quantitative mass spectrometry, we identify S676 as a Plk1-specific phosphorylation site on BubR1. Furthermore, using a phospho-specific antibody, we show that this site is phosphorylated during prometaphase, but dephosphorylated at metaphase upon establishment of tension between sister chromatids. These findings describe the first in vivo verified phosphorylation site for human BubR1, identify Plk1 as the kinase responsible for causing the characteristic mitotic BubR1 upshift, and attribute a KT-specific function to the hyperphosphorylated form of BubR1 in the stabilization of KT-MT interactions.