Triple-negative breast cancer (TNBC) is characterized by fast growth, high metastasis, high invasion, and a lack of therapeutic targets. Mitosis and metastasis of TNBC cells are two important biological behaviors in TNBC malignant progression. It is well known that the long noncoding RNAAFAP1-AS1plays a crucial role in various tumors, but whetherAFAP1-AS1is involved in the mitosis of TNBC cells remains unknown. In this study, we investigated the functional mechanism of AFAP1-AS1 in targeting Polo-like Kinase 1 (PLK1) activation and participating in mitosis of TNBC cells. We detected the expression ofAFAP1-AS1in the TNBC patient cohort and primary cells byin situhybridization (ISH), northern blot, fluorescentin situhybridization (FISH) and cell nucleus/cytoplasm RNA fraction isolation. High AFAP1-AS1 expression was negatively correlated with overall survival (OS), disease-free survival (DFS), metastasis-free survival (MFS) and recurrence-free survival (RFS) in TNBC patients. We explored the function ofAFAP1-AS1by transwell, apoptosis, immunofluorescence (IF) and patient-derived xenograft (PDX) modelsin vitroandin vivo. We found thatAFAP1-AS1promoted TNBC primary cell survival by inhibiting mitotic catastrophe and increased TNBC primary cell growth, migration and invasion. Mechanistically,AFAP1-AS1activated phosphorylation of the mitosis-associated kinase PLK1 protein. Elevated levels ofAFAP1-AS1in TNBC primary cells increased PLK1 pathway downstream gene expression, such as CDC25C, CDK1, BUB1 and TTK. More importantly,AFAP1-AS1increased lung metastases in a mouse metastasis model. Taken together,AFAP1-AS1functions as an oncogene that activates the PLK1 signaling pathway.AFAP1-AS1could be used as a potential prognostic marker and therapeutic target for TNBC.