The present study was conducted to identify the major heparin-binding proteins (HBPs) in the seminal plasma from tropically adapted Morada Nova rams, raised in the Northeast of Brazil. Seminal plasma samples from six adult, reproductively sound rams were collected and subjected to heparin affinity chromatography. HBPs were separated by one-dimensional SDS-PAGE and the main 13 bands were excised from the gel, digested with trypsin and identified by tandem mass spectrometry. Based on the integration of the area under the chromatographic peaks, the vast majority of proteins in the ram seminal plasma (83.5%) matched to those without affinity for heparin. However, only 16.5% of these proteins effectively bind to heparin. HBPs identified in this study include clusterin, Bodhesin 2, matrix metalloproteinase 2, beta mannosidase and dentin matrix acidic phosphoprotein 1, among others. Given the putative biochemical characteristics of HBPs, we suggest that heparin-binding proteins of the ram seminal plasma modulate several aspects of the reproductive events, such as sperm capacitation, formation of the oviduct reservoir, sperm–egg binding, and protection against microorganisms and oxidative stress. In contrast to what has been reported for other species, binder of sperm proteins (BSP) expressed in ram seminal plasma were not detected among HBPs. These findings were confirmed by both mass spectrometry and Western blot. These findings are novel and raise questions about BSPs ability to capacitate ram sperm, as they function in other mammals, such as the bovine. The approach used in the present study also allowed the identification of proteins never reported before in the ram seminal plasma, such as dentin matrix acidic phosphoprotein, cathelicidin and lactoperoxidase. Considering the importance of heparin as a binding partner in reproductive events, our findings give new insights on the roles of seminal plasma proteins in the ram.