Background Breast cancer, particularly the triple-negative breast cancer (TNBC) subtype, remains a significant clinical challenge due to its resistance to standard chemotherapy and high recurrence rate. In this study, we explored the role of Sterile Alpha Motif Domain-Containing 5 (SAMD5) as a potential regulatory partner with the c-Myc oncogenic signaling pathway in breast cancer. Materials and methods Functional assays were conducted to investigate the effects of SAMD5 overexpression on cell viability, colony formation, and invasive behavior in TNBC cell lines. This study further assessed the expression levels of proliferation and invasion markers, including Ki67 (a marker for cell proliferation), Matrix Metalloproteinase-2 (MMP2), and Matrix Metalloproteinase-9 (MMP9). Mechanistic analyses identified a negative correlation between SAMD5 and Polo-like Kinase 1 (PLK1), a gene frequently overexpressed in breast cancer, particularly in TNBC. The effects of PLK1 knockdown on cell viability, colony formation, and invasion were observed, along with the impact of PLK1 overexpression on SAMD5's inhibitory activity. In vivo studies were performed using a xenograft tumor model in nude mice to evaluate the impact of SAMD5 overexpression on tumor weight and volume. Results SAMD5 overexpression significantly reduced cell viability, colony formation, and invasion in TNBC cells, and downregulated key proteins in the c-Myc signaling pathway, including c-Myc itself, β-catenin, Cyclin-Dependent Kinase 4 (CDK4), Cyclin-Dependent Kinase 6 (CDK6), and Cyclin D1. PLK1 overexpression was found to counteract SAMD5's inhibitory effects. In vivo experiments demonstrated that SAMD5 overexpression led to a marked reduction in tumor weight and volume, effects that were partially reversed by PLK1 overexpression. Conclusions SAMD5 acts as a tumor suppressor in breast cancer, particularly in TNBC, by inhibiting critical cellular processes and downregulating the c-Myc signaling pathway. This effect appears to be mediated, in part, through its negative association with PLK1. Targeting the SAMD5/PLK1 axis offers a promising therapeutic strategy for addressing aggressive breast cancers.