Clip domain serine proteases (clip-SPs) play critical roles in various immune responses in arthropods, such as hemolymph coagulation, antimicrobial peptide (AMP) synthesis, cell adhesion and melanization. In the present study, we report the molecular and functional characterization of a clip domain serine protease (PtcSP2) from the swimming crab Portunus trituberculatus. The N-terminal clip domain and the C-terminal SP-like domain of PtcSP2 were expressed in Escherichia coli system, and assayed for their activities. Sequence similarity and phylogenetic analysis revealed that PtcSP2 may belong to the chymotrypsin family, which was confirmed by protease activity assay of the recombinant SP-like domain. The clip domain of PtcSP2 exhibited strong antibacterial activity and microbial-binding activity, suggesting the potential role in immune defense and recognition. Knockdown of PtcSP2 by RNA interference could significantly reduce PtcSP2 transcript levels, but neither decrease the total phenoloxidase (PO) activity in crab nor significantly alter the expression levels of serine protease inhibitors PtPLC and PtSerpin. These results indicate that PtcSP2 is not involved in the proPO system. However, suppression of PtcSP2 led to a significant change in the expression of AMP genes PtALFs and PtCrustin but not PtALF5. All these findings suggest that PtcSP2 is a multifunctional chymotrypsin-like serine protease and may participate in crab innate immunity by its antibacterial activity, immune recognition or regulation of AMP expression.