Intervertebral disc (IVD) is often the cause of low back pain. Degeneration occurs with age and is accompanied by extracellular matrix (ECM) depletion, culminating in nucleus pulpous (NP) extrusion and IVD destruction. Although the changes that occur with ageing in the IVD have been under study, not much attention has been given to ECM remodelling during normal development. Therefore, a thorough study of ECM composition and a comprehensive view on how this microenvironment is affected in normal ageing conditions is needed, to better understand the processes involved in IVD development and in age-associated degeneration. We have compared the NP matrisome of bovine IVDs from foetus, young and old animals by quantitative iTRAQ LC-MS/MS. Protein expression levels of the most interesting candidates were validated by Western Blot analysis. In total, 161 bovine proteins were identified. Of these, 77 molecules were common to the three different age groups, of which 36 defined the NP matrisome. Differential expression levels obtained for Collagen Type XI, XII, XIV, Fibronectin and Prolargin were independently confirmed. Herein, we demonstrate a shift in NP matrisome signatures that occurs in healthy bovine IVDs with development and ageing. Thus, this study provides insight into factors that may explain age-associated IVD degeneration, and which are putative targets for therapy. It also highlights key molecules, characteristic of early developmental stages, which constitute potential effectors in IVD regeneration.