The present study is focused on the proteome of reproductive tract fluids from tropically-adapted Santa Ines rams. Seminal plasma, cauda epididymal (CEF) and vesicular gland fluid (VGF) proteins were analyzed by 2-D electrophoresis and mass spectrometry. Seminal plasma maps contained 302±16 spots, within the 4–7 pH range. From these maps, 73 spots were identified, corresponding to 41 proteins. Ram Seminal Vesicle Proteins (RSVP) 14 and 22kDa and bodhesins 1 and 2 represented the most abundant seminal components. Other seminal proteins included clusterin, angiotensin-converting enzyme, matrix metalloproteinase-2, tissue-inhibitor of metalloproteinase-2, plasma glutamate carboxypeptidase, albumin, lactoferrin, alpha enolase, peroxiredoxin, leucine aminopeptidase, β-galactosidase, among others. Later, seminal plasma gels were run within narrow pH intervals (3.9–5.1; 4.7–5.9; 5.5–6.7), allowing the additional identification of 21 proteins not detected in 4–7 pH maps. Major proteins of CEF and VGF were albumin and transferrin, and RSVPs, respectively. Western blots confirmed that RSVPs were mainly present in VGF while bodhesins, in VGF and CEF. Based on RT-PCR, RSVP and bodhesin genes were primarily expressed in the vesicular glands. In summary, the reproductive tract fluids of Brazilian hairy rams contain several categories of proteins, with potential roles in sperm protection, capacitation, acrosome reaction and sperm–oocyte interaction. This article is part of a Special Issue entitled: Farm animal proteomics. Display omitted ► We evaluated the proteome of reproductive fluids from hair Santa Ines rams. ► Attributes of seminal plasma (SP), epididymal and seminal vesicle proteins are diverse. ► Major SP proteins, Binder of Sperm Proteins and spermadhesins, bind to sperm. ► Seminal plasma gels that run with narrow pH ranges allow identification of more proteins. ► Expression of certain SP proteins related to the reproductive development of rams.