Transplantation is currently the best treatment option for end-stage renal disease (ESRD) patients. However, acute rejection (AR) is the main source of failure in renal transplantation. The current gold standard for diagnosis of AR involves renal biopsy, but it is invasive, time consuming, costly and inconvenient. Sensitive and less invasive detection of AR episodes in renal transplant patients is essential to preserve the allograft function. Here, we applied isobaric tags for relative and absolute quantisation (iTRAQ) mass spectrometry to analyse serum protein expression in AR patients and healthy controls. Overall, 1399 proteins were identified. Using a cut-off of Q<0.05 and a fold change > 1.2 for the expressed variation, 109 proteins showed distinct differential expression between the AR and control groups, of whom 72 were upregulated and 37 were downregulated. Several proteins, such as properdin, keratin 1, lipoprotein (a) and vitamin D-binding protein, may play roles in the pathogenesis of AR. This study focused on iTRAQ-based proteomic profiling of serum samples in AR. Insights from our work might help advance our understanding of the molecular mechanisms of AR and identify potential novel biomarkers of AR for further characterization.