Background and Aims Triple negative breast cancer (TNBC) is more prone to invasion and metastasis, with a high degree of malignancy. Previous study of the authors’ team found the low expression of Fat atypical cadherin 4 (FAT4) in TNBC tissue and its prognostic relevance. Thus, this study was conducted to further investigate the effects of FAT4 on the biological behavior of TNBC cells and the associated mechanism.Methods The expression levels of FAT4 in normal mammary epithelial cell line (MCF-10A) and different TNBC cell lines (BT-549, MDA-MB-231, MDA-MB-468, and MDA-MB-436) were detected by qRT-PCR and Western blot, respectively. Then, the suitable TNBC cell lines were selected, and were transfected with FAT4-shRNA (FAT4 knockdown group) or scrambled sequences (negative control group), using untransfected TNBC cells as blank control group. In these cells, the changes in proliferation ability, apoptosis rate, and invasion/metastasis ability were determined by CCK-8 assay, flow cytometry and Transwell chamber assay, respectively. Meanwhile, the changes in YAP protein, a downstream target of the Hippo signaling pathway, as well as the epithelial mesenchymal transformation (EMT)-related proteins were also examined by Western blot analysis.Results Compared with normal mammary epithelial cell line, the mRNA and protein expression levels of FAT4 in all the studied TNBC cell lines were decreased with varying degrees (all P<0.05). The BT-549 and MDA-MB-436 cells were selected for functional experiments. In FAT4 knockdown group compared with blank control group of either the BT-549 or MDA-MB-436 cells, the proliferative abilities were significantly increased at 24, 48, and 72 h after transfection, the apoptosis rate was significantly decreased, and the invasion/metastasis ability was significantly enhanced (all P<0.05); the expression level of YAP protein showed no significant change (P>0.05), but the level of phosphorylated YAP (p-YAP) was significantly decreased, and the level of epithelial marker E-cadherin was significantly decreased and mesenchymal marker N-cadherin was significantly increased (all P<0.05); the differences in all above parameters showed no statistical significance between negative control group and blank control group (all P>0.05).Conclusion The expression of FAT4 is generally down-regulated in TNBC cells. The FAT4 down-regulation can enhance the proliferation and invasion/migration abilities and weaken the apoptosis of TNBC cells, and the mechanism may be probably associated the reduced phosphorylation of YAP protein in downstream Hippo signaling pathway after FAT4 down-regulation, and thereby promoting the EMT process.