To evaluate anti-tumor effects of parabiosis followed by breast tumor cells re-challenge, breast tumor cells were injected to fourth mammary gland in one mouse of parabiosis mice. After 28 days, tumor cells were re-challenged to the other mice without tumor from separated parabiosis mice (Separant-tumor), and tumor growth was observed for 14 days. As positive control as only tumor cells injected mice (Tumor) and naïve mice (Naïve) as negative control were used. To screen out candidates of anti-tumor protein in serum of mice, liquid chromatography–mass spectrometry (LC-MS) was performed.