Ovine pulmonary adenocarcinoma (OPA) is a retrovirus-induced tumour that has been considered a model for human lung cancer research. The present study aimed to identify a protein marker associated with OPA growth, using proteomic methods and immunohistochemistry.Protein lysates obtained from frozen lung tissues of OPA-affected sheep (n = 5) and non-neoplastic lungs (n = 4) were submitted for analysis by two-dimensional electrophoresis (2DE) using pH range 3–10 in isoelectric focusing. After 2DE gel comparison, selected gel spots presenting over two-fold overexpression in OPA were submitted for protein identification using liquid chromatography–tandem mass spectrometry. Next, immunohistochemical analysis was performed using an antibody against triosephosphate isomerase 1 (TPI-1), which was a protein identified in one of the 2DE spots. The material consisted of paraffin wax-embedded tissues of OPA-affected sheep (n = 5), non-neoplastic lungs with inflammatory lesions (n = 5) and lungs with no visible lesions (n = 5). As a result of the proteomic analysis, TPI-1 was identified as overexpressed in OPA 2DE gels. IHC analysis revealed moderate to strong TPI-1 nuclear labelling in epithelial cells in bronchioli as well as in lung macrophages in all the sections. In OPA sections, the tumour cells had moderate cytoplasmic and moderate to strong nuclear labelling. In all cases, BALT tissue showed mild cytoplasmic labelling. The proteomic methods allowed identification of TPI-1, a glycolytic enzyme involved in neoplastic growth. IHC results confirmed that the overexpression of TPI-1 was tumour-derived, while TPI-1 expression in macrophages might be associated with activation and motility.