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To identify acute renal allograft rejection biomarkers in human serum, two-dimensional differential in-gel electrophoresis (2-D DIGE) and reversed phase high-performance liquid chromatog-raphy (RP-HPLC) followed by electrospray ionization mass spectrometry (ESI-MS) were used. Serum samples from renal allograft patients and normal volunteers were divided into three groups: acute rejec-tion (AR), stable renal function (SRF) and normal volunteer (N). Serum samples were firstly processed using Multiple Affinity Removal Column to selectively remove the highest abundance proteins. Differ-entially expressed proteins were analyzed using 2-D DIGE. These differential protein spots were ex-cised, digested by trypsin, and identified by RP-HPLC-ESI/MS. Twenty-two differentially expressed proteins were identified in serum from AR group. These proteins included complement C9 precursor,apolipoprotein A-Ⅳ precursor, vitamin D-binding protein precursor, beta-2-glycoprotein 1 precursor,etc. Vitamin D-binding protein, one of these proteins, was confirmed by ELISA in the independent set of serum samples. In conclusion, the differentially expressed proteins as serum biomarker candidates may provide the basis of acute rejection noninvasive diagnosis. Confirmed vitamin D-binding protein may be one of serum biomarkers of acute rejection. Furthermore, it may provide great insights into un-derstanding the mechanisms and potential treatment strategy of acute rejection.