MicroRNA (miRNA) deregulation has been previously linked to the initiation and development of breast cancer. Although miR-99a is aberrantly expressed in many types of cancers, including breast cancer, the serum miR-99a expression level in breast cancer and its clinical significance remains unknown. Blood samples were obtained from 72 patients with breast cancer and 40 healthy volunteers, and subjected to real-time polymerase chain reaction to evaluate the level of expression of serum miR-99a in the study participants. Furthermore, we investigated the association between serum miR-99a and the clinical outcome of breast cancer. Serum miR-99a expression was significantly downregulated in patients with breast cancer, compared to that in healthy controls (P < 0.01). Moreover, the serum miR-99a was correlated with various clinical parameters of breast cancer, including lymph node metastasis (P = 0.0194), distant metastasis (P = 0.0037), Ki67 intensity (P = 0.0164), TNM stage (P = 0.0096), and histological grade (P = 0.0051) of cancer. Additionally, breast cancer patients displaying lower miR-99a levels showed poorer overall survival rates (P = 0.0411). The serum miR-99a level was also found to be an independent risk factor for breast cancer (hazard ratio = 3.176, 95% confidence interval = 1.543-7.360, P = 0.023). Our data indicated that serum miR-99a expression was downregulated in breast cancer patients; moreover, this downregulation was associated with poor prognosis, suggesting that serum miR-99a could function as a tumor suppressor in breast cancer.