Prefoldin1 (PFDN1), a molecular chaperone, is essential for stabilizing cytoskeletal proteins like actin and tubulin, supporting cellular processes such as survival, migration, and cell cycling. Recent evidence suggests that PFDN1 also influences key cancer-related signaling pathways. However, the complete mechanisms involved and the downstream genes implicated in such action remain relatively undiscovered. This study investigated the effects of PFDN1 silencing on cellular processes and gene expression in triple-negative breast cancer (TNBC) cells, focusing on its potential as a therapeutic target. MDA-MB-231 cells, a TNBC model, were transfected with PFDN1-targeting siRNA to knock down PFDN1 expression. The effects of PFDN1 silencing were assessed through various assays, including phase contrast and scanning electron microscopy (SEM) for morphological changes, colony formation and wound healing assays for proliferation and migration, and flow cytometry for cell cycle and apoptosis analysis. Gene expression changes were evaluated using a qRT-PCR array targeting 84 genes involved in cancer progression. PFDN1 silencing resulted in a 54.8% reduction in PFDN1 protein levels (p <
0.0001). Morphological analysis revealed cytoplasmic shrinkage, chromatin condensation, roughened membranes, and microvilli loss, consistent with apoptotic changes. Colony formation assays showed a 10.33% reduction in colony number and size (p <
0.05) in PFDN1-silenced cells. Migration was significantly impaired, with reduced wound closure observed in wound healing assays (p <
0.01). Flow cytometry revealed a G2/M phase arrest (p <
0.05) and increased early apoptotic populations (20.93% vs. 5.42% in controls, p <
0.01). Gene expression analysis showed downregulation of genes associated with angiogenesis (KDR, TEK), EMT (FOXC2, SNAI1), and hypoxia signaling (CA9, EPO), while proapoptotic genes, such as FASLG, were upregulated. This study highlights the critical role of PFDN1 in TNBC progression, demonstrating that its silencing disrupts survival, migration, cell cycling, and apoptosis pathways. PFDN1 knockdown also significantly alters the expression of key cancer-related genes, further impairing angiogenesis, EMT, and hypoxia adaptation. These findings suggest that targeting PFDN1 could be a promising therapeutic strategy for TNBC, warranting further investigation in preclinical models.