Actinobacillus seminis is a gram-negative bacterium of the Pasteurellaceae family that is involved in ovine epididymitis. Looking for a protein specific to this species, we determined the protein profile of subcellular fractions of A. seminis (American Type Culture Collection number 15768): proteins from the outer membrane (OMPs), inner membrane (IMPs), and cytoplasm (CPs). These profiles provide the first data, to our knowledge, regarding subcellular fractions of A. seminis. In the OMP fraction, we identified a protein with a molecular mass of 75 kDa that proved to be immunogenic and apparently specific for A. seminis. This conclusion was based on the reaction of hyperimmune serum of rabbits inoculated with whole cells of A. seminis that was tested against sonicated complete cells of reference strains and field isolates of Brucella ovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. No protein of these bacteria cross-reacted with the 75-kDa protein of A. seminis. Furthermore, when each type of hyperimmune serum was tested against the sonicated cells and each of the subcellular fractions of A. seminis, it did not recognize the A. seminis 75-kDa protein. We also isolated and identified this protein in microvesicles released to the culture supernatant. The results suggest that the 75-kDa protein could be used to establish a diagnostic test specific for ovine epididymitis caused by A. seminis.