The effect of 100 nM sodium selenite supplementation was studied on LNCaP cells by a proteomic approach, on ProteomeLab PF 2D platform. Proteins were separated by liquid phase bi-dimensional chromatography and analyzed by pair-wise alignment of peaks to detect those differentially expressed. Differential expression threshold was set at a twice difference level and proteins matching this criterion were identified by MALDI-TOF and confirmed by ESI-ion trap MS/MS. Not all differentially expressed proteins found by PF 2D could be identified by MS analysis, the sensitivity of which emerging as the limiting factor. Thus, only the most abundant proteins, differently expressed following selenium supplementation, were identified. We positively showed an increase of expression of thioredoxin reductase 1, enolase 1, phosphoglycerate mutase 1, glyceraldehyde-3-phosphate dehydrogenase, heterogeneous nuclear ribonucleoprotein A2/B1, isoform A2, Ras-GTPase-activating protein SH3-domain-binding protein and Keratin 18 and a decrease of expression of peroxiredoxin 1 and heat shock protein 70, protein 8, isoform 1. Results are consistent, at least in part, with the less oxidant environment brought about by the synthesis of Se-dependent peroxidases, keeping low the steady-state concentration of hydrogen peroxide.