Abstract Background Sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid mediator, has been implicated as a key regulatory molecule in cancer through its ability to promote cell proliferation, migration, angiogenesis and lymphangiogenesis. Previous studies suggested that S1P produced by sphingosine kinase 1 (SphK1) in breast cancer plays important roles in progression of disease and metastasis. However, the associations between S1P and clinical parameters in human breast cancer have not been well investigated to date. Materials and Methods We determined levels of S1P and other sphingolipids in breast cancer tissue by electrospray ionization-tandem mass spectrometry. Associations between S1P levels and clinicopathological features of the tumors were analyzed. Expression of phospho-SphK1 (pSphK1) in breast cancer tissues was determined by immunohistochemical scoring. Results Levels of S1P in breast cancer tissues were significantly higher in patients with high white blood cell count in the blood than patients without. S1P levels were lower in patients with HER2 overexpression/amplification than patients without. Further, cancer tissues with high pSphK1 expression showed significantly higher levels of S1P than cancer tissues without. Finally, patients with lymph node metastasis showed significantly higher levels of S1P in tumor tissues than patients with negative nodes. Conclusions To our knowledge, this is the first study to demonstrate that high expression of pSphK1 is associated with higher levels of S1P, which in turn is associated with lymphatic metastasis in breast cancer.