Survivin is an essential chromosomal passenger protein whose function remains unclear. Here, we have used RNA interference to specifically repress Survivin in cultured HeLa cells. Immunoblot analysis showed that Survivin was no longer detectable in cultures 60 hours after transfection with Survivin-specific siRNA. Live cell analysis showed that many Survivin-depleted cells were delayed in mitosis, and immunofluorescence analysis of fixed specimens revealed that Survivin-depleted cells accumulated in prometaphase with misaligned chromosomes. The chromosomal passenger proteins, INCENP and Aurora-B, which can interact directly with Survivin, were absent from the centromeres of Survivin-depleted cells. These data contribute to the emerging picture that Survivin operates together with INCENP and Aurora-B to perform its mitotic duties. Some Survivin-depleted cells eventually exited mitosis without completing cytokinesis. This resulted in a gradual increase in the percentage of multinucleated cells in the culture. Time-lapse imaging of synchronized cultures revealed that control and Survivin-depleted cells arrested in mitosis in the presence of nocodazole; however, the latter failed to arrest in mitosis when treated with taxol. Immunofluorescence studies revealed that Survivin-depleted cells were unable to stably maintain BubR1 at the kinetochores in the presence of either taxol or nocodazole. Our data reveal that Survivin is not required for the spindle assembly checkpoint when it is activated by the loss of microtubules. However, Survivin is required for the maintenance of the checkpoint when it is activated by taxol, which is generally thought to cause a loss of spindle tension.