Progesterone secreted by the corpus luteum (CL) is critical for in-utero embryo survival and development, while CL proteins are key regulatory factors during the luteal phase. We therefore characterised protein expression patterns in ovine CLs of pregnancy (days 12, 16 and 20) compared with those of controls, CLs of oestrous cycle (days 12 and 16), using 2DE gel-based proteomics. Proteins in 24 significantly altered spots were identified by tandem mass spectroscopy. At the time of embryo implantation (day 16), 77 spots were up-regulated and 101 spots were down-regulated in CLs of pregnancy compared with regressed CLs. VIM, LMNA, SOD2, IDH1, ANXA1 and TUFM altered during CL regression while GSTA1, APOA1, MX1, OAT and ECHS1 tended to be altered during CL maintenance. BLVRB, FDXR, GNB2 and UQCRC1, showed divergent expression during CL regression and maintenance. The expression of two representative proteins, SOD2 and BLVRB by Western blot was increased in CLs of non-pregnant ewes on day 16 compared to day 12. Manganese containing SOD (Mn-SOD or SOD2) and biliverdin reductase B (BLVRB) were localised in the large and small luteal cells and endothelial cells of CLs over peri-implantation periods. 2DE gel and mass spectrometry have been used, for the first time, to study ovine CL function. We have identified proteins involved in key pathways, including oxidative stress, steroidogenesis, signal transduction and apoptosis, which have not previously been associated with changes occurring in the CL during the peri-implantation period. These proteins are most likely involved with mechanisms allowing the CL to produce progesterone during early pregnancy.