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Objective: To verify the role of the newly identified mitotic regulator candidate pre-mRNA processing factor 19 (Prp19) in mitosis and to clarify its underlying mechanism., Methods: FACS analyses with propidium iodide (PI) staining were performed to evaluate the effect of Prp19 knockdown on cell cycle distribution. To further clarify the role of Prp19 in mitosis, the effect of Prp19 depletion was monitored by time-lapse imaging of HeLa/GFP-H2B cells. Cold treatment experiment was used to examine the effect of Prp19 knockdown on the attachment of microtubules and kinetochores. To evaluate the effect of Prp19 knockdown on cell apoptosis, the control and Prp19-knockdown cells were analyzed by FACS with annexin V-FITC/-PI double staining. Furthermore, Western blot analysis of cleaved caspase-3 and PARP was also performed., Results: Prp19 knockdown causesd mitotic arrest. Time-lapse imaging analysis showed that depletion of Prp19 in HeLa cells results in prometaphase arrest and chromosome misalignment. Cold treatment experiment showed that attachment between kinetochore and microtubule was impaired by Prp19 knockdown. Moreover, the depletion of Prp19 leaded to cell apoptosis in cancer cells., Conclusion: Prp19 is a key regulator of mitotic progression, and its inhibition may provide a new strategy for anti-cancer therapy.