MDC1 and BRIT1 have been shown to function as key regulators in response to DNA damage. However, their roles in centrosomal regulation haven’t been elucidated. In this study, we demonstrated the novel functions of these two molecules in regulating centrosome duplication and mitosis. We found that MDC1 and BRIT1 were integral components of the centrosome that colocalize with γ-tubulin. Depletion of either protein led to centrosome amplification. However, the mechanisms that allow them to maintain centrosome integrity are different. MDC1-depleted cells exhibited centrosome overduplication, leading to multipolar mitosis, chromosome missegregation, and aneuploidy, whereas BRIT1 depletion led to misaligned spindles and/or lagging chromosomes with defective spindle checkpoint activation that resulted in defective cytokinesis and polyploidy. We further illustrated that both MDC1 and BRIT1 were negative regulators of Aurora A and Plk1, two centrosomal kinases involved in centrosome maturation and spindle assembly. Moreover, the levels of MDC1 and BRIT1 inversely correlated with centrosome amplification, defective mitosis, and cancer metastasis in human breast cancer. Together, MDC1 and BRIT1 may function as tumor-suppressor genes, at least in part by orchestrating proper centrosome duplication and mitotic spindle assembly.