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Subclinical hypocalcaemia (SH) is an important metabolic disease in dairy cows that has a serious impact on production performance. The objective of this study was to investigate novel aspects of pathogenesis using proteomics technology to identify proteins that are differentially expressed in diseased and healthy animals. Dairy cows were divided into an SH group (T, n  = 10) and a control group (C, n  = 10) based on plasma calcium concentration. A total of 398 differentially expressed proteins were identified, of which 265 proteins were overlapped in the two parallel experiments. Of these, 24 differentially expressed proteins were statistically significant. Gene Ontology analysis yielded 74 annotations, including 7 cellular component, 55 biological process and 12 molecular function categories. Bioinformatics analysis indicated that calcium regulation, immune and inflammatory response, blood coagulation and complement pathway were all related to SH. Our iTRAQ/LC-MS/MS (isobaric tags for relative and absolute quantification/liquid chromatography-mass spectrometry/mass spectrometry) approach proved highly effective for plasma protein profiling of dairy cows with SH, and the results pave the way for further studies in this area.