Serine proteinases (SPs) participate in various biological processes and play vital role in immunity. In this study, we investigated the function of PmClipSP2 from shrimp Penaeus monodon in defense against bacterial infection. PmClipSP2 was identified as a clip-domain SP and its mRNA increased in response to infection with Vibrio harveyi. PmClipSP2-knockdown shrimp displayed a significantly reduced phenoloxidase (PO) activity and increased susceptibility to V. harveyi infection. Injection of LPS and/or β-1,3-glucan induced a dose-dependent mortality and a significant decrease in the number of total hemocytes, with clear morphological changes in the hemocyte surface, of the PmClipSP2-knockdown shrimp. Recombinant PmClipSP2 was shown to bind to LPS and β-1,3-glucan and to activate PO activity. These results reveal that PmClipSP2 acts as a pattern-recognition protein, binding to microbial polysaccharides and likely activating the proPO system, whilst it may play an essential role in the hemocyte homeostasis by scavenging LPS and neutralizing its toxicity.