Adequate lymph node evaluation is recommended for optimal staging in patients with malignant neoplasms including breast cancer. However, the role of negative lymph nodes (LNs) remains unclear in breast cancer according to N substage (N1, N2, and N3). In this study, for the first time, we analyzed the prognostic significance of negative LNs in breast cancer patients. A critical relationship was observed between negative LN count and survival, independent of patient characteristics and other related molecular variables including estrogen receptor (PR) status, progesterone receptor (ER) status, human epidermal growth factor receptor 2 (HER2) status, depth of tumor invasion and degree of differentiation. This research is of great importance in providing more information about the prognosis of breast cancer by statistical analysis of negative lymph nodes and can serve as a useful supplement to the current pathological system.