The yeast separase proteins Esp1 and Cut1 are required for loss of sister chromatid cohesion that occurs at the moment of anaphase onset. Circumstantial evidence has linked human separase to centromere separation at anaphase, but a direct test that the role of this enzyme is functionally conserved with the yeast proteins is lacking. Here we describe the effects of separase depletion from human cells using RNA interference. Surprisingly, HeLa cells lacking separase are delayed or arrest at the G2-M phase transition. This arrest is not likely due to the activation of a known checkpoint control, but may be a result of a failure to construct a mitotic chromosome. Without separase, cells also have a prolonged prometaphase, perhaps resulting from defects in spindle assembly or dynamics. In cells that reach mitosis, sister arm resolution and separation are perturbed, whereas in anaphase cells sister centromeres do appear to separate. These data indicate that separase function is not restricted to anaphase initiation and that its role in promoting loss of sister chromatid cohesion might be preferentially at arms but not centromeres.