Contents We have already shown that RSVP14 and RSVP20, two ram seminal plasma (SP) proteins postulated to be involved in sperm capacitation and gamete interaction can protect spermatozoa against cold‐shock. In this study, we use two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) for the analysis of SP proteins of Rasa Aragonesa rams, using enhanced protein solubilization in the presence of tributyl phosphine (TBP) and a polyacrylamide linear gradient gel with a narrow pH range (4–7). The image analysis of the 2D map detected 195 protein spots, with isoelectric points (pIs) ranging from 4.5 to 6.6, and molecular weight ( M r ) from 11.7 to 90.4. Staining of 2D gels with Pro‐Q Emerald 300 Glycoprotein Stain revealed that most significant proteins in ram SP are glycosylated. The removing of protein N‐linked oligosaccharides improved the gel resolution. 2D‐PAGE analysis of the whole fraction 6 (F6) separated from ram SP by exclusion chromatography showed six main protein spots, four ( a , b , c , d ) in the 14 kDa and two ( e , f ) in the 20 kDa region. Western‐blot analyses indicated that the anti‐P14 antibody recognized four spots on the SP map, 4, 5, 6 and 7, that matched with spots a , b , c , d of F6 map. The anti‐P20 antibody recognized spots 13 and 14 of SP map that corresponded to spots e , f of F6 map. The deduced sequences by de novo sequencing evidenced that protein spots 7 and 13 have significant similarities to BSP family, while protein spots 4 and 14 did not appear to be homologous with any reported protein in the current mammalian Proteinbank databases.