Streptococcus suis (S. suis) is a zoonotic pathogen causing severe streptococcal disease worldwide. S. suis infections in pigs and humans are frequently associated with the virulent S. suis serotype 2 (SS2). Though various virulence factors of S. suis have been proposed, most of them were not essentially accounted for in the experimental infections. In the present study, we compared the peptidomes of highly virulent SS2 and SS14 in humans, the swine causative serotypes SS7 and SS9, and the rarely reported serotypes SS25 and SS27, and they were cultured in a specified culture medium containing whole blood to simulate their natural host environment. LC-MS/MS could identify 22 unique peptides expressed in the six S. suis serotypes. Under the host-simulated environment, peptides from the ABC-type phosphate transport system (SSU05_1106) and 30S ribosomal protein S2 (rpsB) were detected in the peptidome of virulent SS2 and SS14. Therefore, we suggest that these two proteins or their derived peptides might be involved in the survival of S. suis when simulated with a blood environment.