Abstract Cell division cycle 20 (Cdc20) is the main activator of the anaphase-promoting complex (APC/C) and is essential in mitosis regulation. Recently, several papers reported that Cdc20 is highly conserved in various human cancers such as pancreatic, prostate, and lung cancers yet its relation to breast cancer is unclear. Here, we found that Cdc20 gene was highly expressed in three different TNBC cell lines among the mitosis regulator genes through the National Center for Biotechnology Information (NCBI). High Cdc20 level is positively correlated with metastasis-free patient survival and relapse-free patient survival in breast cancer patient database. We also checked that Cdc20 is highly conserved in all TNBC cell lines compared to other subtypes of breast cancer cell lines and the knockdown of this gene decreased cell growth and invasion in TNBC. Furthermore, we investigated that the mitosis-related small molecule protein kinase inhibitors such as VX-680, ZM447439, BI2536, and apcin also inhibit tumor cell proliferation, migration, and invasion. In conclusion, Cdc20 has an important role in TNBC cell growth and metastasis. Cdc20 might serve as a potential marker of disease progression and as a principal potential therapeutic target. Also, mitosis-related protein kinase inhibitors are additional potential therapy to suppress cancer cell progression and invasion in TNBC effectively. Citation Format: Chaeun Song, SeungBaek Lee. Mitotic inhibitors suppresses tumor formation and metastasis of triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2890.