Background <p>Breast cancer is an important cause of death from malignant tumors in women. This study aims to clarify the regulatory role, molecular mechanism and clinical value of miR-3648 in breast cancer.</p> Methods <p>This study analyzed 126 pairs of clinical samples of breast cancer. The expression of miR-3648 and its potential target SMAD6 was detected by qRT-PCR. Survival outcomes were assessed by Kaplan-Meier analysis and Cox regression model. In the MCF-7 and BT-474 cell lines, function gain and loss experiments were conducted using miR-3648 mimic or inhibitor. Biological behaviors modulated by miR-3648, including cell proliferation, migration and invasion were assessed using CCK-8 and Transwell assays. The target genes of miR-3648 were predicted by bioinformatics, and their relationship was verified by dual-luciferase.</p> Results <p>MiR-3648 was significantly upregulated in breast cancer and correlated with unfavorable clinicopathological features and predicted the survival rate of patients. In vitro experiments revealed that overexpression of miR-3648 promotes the proliferation, migration, and invasion of breast cancer cells, while inhibition of its expression yields opposite effects. Bioinformatics analysis and verification experiments jointly identified SMAD6 as the functional target of miR-3648. SMAD6 and miR-3648 expression was inversely correlated in breast cancer tissues, and SMAD6 knockdown partially reversed the effects of miR-3648 inhibition.</p> Conclusions <p>miR-3648 exerts its role in promoting the progression of breast cancer by directly targeting and inhibiting SMAD6, and can serve as a potential biomarker for evaluating the prognosis of patients.</p>