Side population (SP) cells have stem cell-like properties with a capacity for self-renewal and are resistant to chemotherapy and radiotherapy. Therefore the presence of SP cells in human breast cancer probably has prognostic value. Objective: To investigate the characteristics of SP cells and identify the relationship between the SP cells levels and clinico-pathological parameters of the breast tumor and disease-free survival (DFS) in breast cancer patients. Materials and Methods: A total of 122 eligible breast cancer patients were consecutively recruited from January 1, 2006 to December 31, 2007 at Yunnan Tumor Hospital. All eligible subjects received conventional treatment and were followed up for seven years. Predictors of recurrence and/or metastasis and DFS were analyzed using Cox regression analysis. Human breast cancer cells were also obtained from fresh human breast cancer tissue and cultured by the nucleic acid dye Hoechst33342 with Verapami. Flow cytometry (FCM) was employed to isolate the cells of SP and non-SP types. Results: In this study, SP cells were identified using flow cytometric analysis with Hoechst 33342 dye efflux. Adjusted for age, tumor size, lymph nodal status, histological grade, the Cox model showed a higher risk of recurrence and/or metastasis positively associated with the SP cell level (1.75, 1.02-2.98), as well as with axillary lymph node metastasis (2.99, 1.76-5.09), pathology invasiveness type (1.7, 1.14-2.55), and tumor volume doubling time (TVDT) (1.54, 1.01-2.36). Conclusions: The SP cell level is independently associated with tumor progression and clinical outcome after controlling for other pathological factors. The axillary lymph node status, TVDT and the status of non-invasive or invasive tumor independently predict the prognosis of breast cancer.