Sister chromatids in mammalian cells remain attached mostly at their centromeres at metaphase because of the loss of cohesion along chromosome arms in prophase. Here, we report that Bub1 retains centromeric cohesion in mitosis of human cells. Depletion of Bub1 or Shugoshin (Sgo1) in HeLa cells by RNA interference causes massive missegregation of sister chromatids that originates at centromeres. Surprisingly, loss of chromatid cohesion in Bub1 and Sgo1 RNA-interference cells does not appear to require the full activation of separase but, instead, triggers a mitotic arrest that depends on Mad2 and Aurora B. Bub1 maintains the steady-state levels and centromeric localization of Sgo1. Therefore, Bub1 protects centromeric cohesion through Shugoshin in mitosis.spindle checkpoint ͉ kinetochore ͉ cohesin