Staphylococcus aureus has one essential type I signal peptidase (SPase), SpsB, which has emerged as a potential target in the search for antibiotics with a new mode of action. In this framework, the biochemical properties of SpsB are described and compared with other previously characterized SPases. Two different substrates have been used to assess the in vitro processing activity of SpsB: (a) a native preprotein substrate immunodominant staphylococcal antigen A and (b) an intramolecularly quenched fluorogenic synthetic peptide based on the sequence of the SceD preprotein of Staphylococcus epidermidis for fluorescence resonance energy transfer-based analysis. Activity testing at different pH showed that the enzyme has an optimum pH of approximately 8. The pH-rate profile revealed apparent pKa values of 6.6 and 8.7. Similar to the other SPases, SpsB undergoes self-cleavage and, although the catalytic serine is retained in the self-cleavage product, a very low residual enzymatic activity remained. In contrast, a truncated derivative of SpsB, which was nine amino acids longer at the N-terminus compared to the self-cleavage product, retained activity. The specificity constants (kcat/Km) of the full-length and the truncated derivative were 1.85 ± 0.13 × 103 M−1·s−1 and 59.4 ± 6.4 M−1·s−1, respectively, as determined using the fluorogenic synthetic peptide substrate. These observations highlight the importance of the amino acids in the transmembrane segment and also those preceding the catalytic serine in the sequence of SpsB. Interestingly, we also found that the activity of the truncated SpsB increased in the presence of a non-ionic detergent.