Cerebrospinal fluid (CSF) is mostly secreted by choroid plexus (CP) and has a unique composition that is different from plasma. The CSF is in direct contact with the brain interstitial fluid, and the composition of the CSF can reflect the biological processes of the brain (1). Identifying CSF biomarkers in neurodegenerative diseases could monitor the functioning of the brain and aid in the diagnosis. However, the CSF protein changes with advancing age in the absence of disease are not fully elucidated. This study aimed to characterize age-related protein changes in ovine CSF by a proteomic approach. The advantage of using sheep in this study is that adequate CSF samples of all ages can be obtained with easier control over gender selection and environmental factors. Unlike humans, sheep do not develop neurodegenerative diseases in ageing (2). Clun Forest strain adult female sheep aged between 1 and 10 year old were anaesthetized with i.v. thiopentone sodium (20 mg.kg−1) and the CSF samples were collected from the cisterna magna (3). All procedures were within the Home Office Scientific procedures Act, 1986 (HMSO, London, UK). Equal volume of CSF samples from seven of each young (1-2 year old), middle aged (3-6 year old), old (7-10 year old) group were pooled. Up to 90 μg of protein from each group were labelled with iTRAQ reagents and were combined. Proteins were fractionated by 2-dimensional high-performance, liquid chromatography. Tryptic peptides in each spot were both identified (using MS/MS fragmentation ions for sequencing) and quantified from iTRAQ reporter ion intensities at m/z 114, 115, 116 and 117. Biomarker validation were performed with immunoassays, such as ELISA and Western immunoblotting. There were 230 peptides analysed by the MS/MS, among which 224 peptides were identified and belonged to 152 proteins. Seven peptides (6 proteins), neuropeptide Y, neuroendocrine protein 7B2, fibrous sheath interacting protein 1, haptoglobin, haemoglobin, IgM were gradually increased, while histone deacetylase was gradually decreased more than one fold following increased age. Glutathione S-transferase was decreased in middle aged CSF for more than one fold compared to both young or old CSF. Serum paraoxonase/arylesterase 1 (PON1) was increased, while nuclear factor of activated T cells was decreased in old CSF more than one fold compared to both young and middle aged CSF. Some of these biomarkers were validated with commercially available ELISA kits and with Western blotting, such as Neuropeptide Y, Neuroendocrine protein 7B2, IgM, haptoglobin, haemoglobin. In conclusion, this study has identified a number of age-related proteins in ovine CSF, which may help us to elucidate the causes for some neurodegenarative diseases, most of which occur at the old age.