As a new-found aquaculture pathogen, Spiroplasma eriocheiris, has resulted in inconceivable economic losses in aquaculture. In the infection of S. eriocheiris, the Procambarus clakii hemocytes have indicated to be major target cells. What was designed to examine in our study is the hemocytes' immune response at the protein levels. Before the pathogen was injected and after 192 h of post-injection, the differential proteomes of the crayfish hemocytes were analyzed immediately by isobaric tags for relative and absolute quantization (iTRAQ) labeling, followed by liquid chromatogramphytandem mass spectrometry (LC-MS/MS). This research had identified a total of 285 differentially expressed proteins. Eighty-three and 202 proteins were up-regulated and down-regulated, respectively, caused by the S. eriocheiris infection. Up-regulated proteins included alpha-2-macroglobulin (α2M), vitellogenin, ferritin, etc. Down-regulated proteins, involved with serine protease, peroxiredoxin 6, 14-3-3-like protein, C-type lectin, cdc42 homolog precursor, etc. The prophenoloxidase-activating system, antimicrobial action involved in the immune responses of P. clarkii is considered to be damaged due to S. eriocheiris infection. The present work could lay the foundation for future research on the proteins related to the susceptibility/resistance of P. clarkii to S. eriocheiris. In addition, it is helpful for our understanding molecular mechanism of disease processes in crayfishes.