Polo-like kinase 1 (PLK1) dynamically changes its localization and plays important roles in proper mitotic progression. In particular, strict control of cytoplasmic PLK1 is needed to prevent mitotic defects. However, the regulation of cytoplasmic PLK1 is not fully understood. In this study, we show that CEP76, a centriolar protein, physically interacts with PLK1 and tightly controls the activation of cytoplasmic PLK1 during mitosis in human cells. We found that removal of centrosomes induced ectopic aggregation of PLK1, which is highly phosphorylated, in the cytoplasm during mitosis. Importantly, a targeted RNA interference (RNAi) screen revealed that depletion of CEP76 resulted in a similar phenotype. In addition, depletion of CEP76 caused defective spindle orientation and mitotic delay. Moreover, the formation of ectopic PLK1 aggregates and defective spindle orientation were significantly suppressed by the inhibition of PLK1 kinase activity. Overall, these results demonstrate that CEP76 suppresses the aberrant activation of cytoplasmic PLK1 for proper mitotic progression.