The aim is to investigate the clinical implications of the Oct-4 and Nestin protein in human breast cancers. A total of 346 cases including 26 fresh and 320 paraffin-embedded tumor tissues were selected for characterizing the frequency of CD44(+)CD24(-) tumor cells by flow cytometry and the differential expression of the stem cell-related genes between CD44(+)CD24(-) and non-CD44(+)CD24(-) tumor cells was analyzed by PCR Array and immunofluorescence. In comparison with the non-CD44(+)CD24(-) tumor cells, the CD44(+)CD24(-), particularly for those with high percentage of Oct-4(+) and Nestin(+), tumor cells had higher tumorigenicity by forming mammospheres in vitro. More importantly, 42 (13.125%) out of 320 tumor tissues were positive for Oct-4 and Nestin staining. Universal analysis and multivariate analysis revealed that the expression of Oct-4 and Nestin was associated significantly with younger age, pathogenic degrees, lymph node metastasis and triple-negative breast cancer independently (P < 0.05) as well as shorter survival (P = 0.001). Oct-4 and Nestin were important regulators of the development of breast cancer, and Oct-4 and Nestin may be used as predictors for the prognosis of breast cancers.