Recently, comprehensive studies were conducted regarding the reproductive development, age at puberty, spermatogenesis and the protein profile in the seminal plasma of Santa Ines rams. Despite the abundant information obtained from these studies regarding these tropically adapted rams in Brazil, it is still unclear how sperm parameters relate to the expression of molecular components of the reproductive tract. In this regard, the present study was conducted to determine if sperm parameters were empirically associated with the seminal plasma proteins described in two-dimensional electrophoresis maps. Seminal plasma proteins were separated by two-dimensional electrophoresis and the respective maps electronically analyzed. Protein spots associated with higher or lower sperm motility were then identified by tandem mass spectrometry. Based on sperm motility, the ejaculates were divided into two groups: those containing up to 80% motile cells (G1; n = 11) and those with more than 80% motile sperm (G2; n = 10). On average, 236 spots per gel were detected. Eleven spots were significantly more intense in gels from animals with ejaculates characterized by higher semen motility scores (G2). The intensity of three other spots was higher in gels from the G1 group. All spots differentially expressed for G1 and G2, were present in at least 90% of the gels. From the 13 spots differentially expressed in G1 and G2, four spots were identified by tandem mass spectrometry. Spots expressed with more intensity in the ejaculates with higher sperm motility (G2) were identified as arylsulfatase A and zinc-alpha-2-glycoprotein. On the other hand, two spots associated with G1 were identified as ram seminal vesicles protein 22 kDa (RSVP-22) and bodhesin-2. Knowledge of these identities represents a crucial step toward the comprehension of how specific seminal plasma proteins are related to sperm motility.