A two-domain Kazal-type serine proteinase inhibitor, SPIPm5, from Penaeus monodon was studied. Its transcript was expressed in all tissues tested including the hemocytes, stomach, gill, lymphoid organ, muscle, intestine and heart albeit less in hepatopancreas and eyestalk. The expression of SPIPm5 gene was also up-regulated by heat stress, white spot syndrome virus (WSSV) infection and yellow head virus (YHV) infection. Injection of recombinant rSPIPm5 protein into normal shrimp to mimic heat stress condition did not have or had little stimulating effect on the expression of other immune genes: crustinPm1, penaeidin3, penaeidin5, Hsp70, SPIPm2 and SPIPm5. Like some other proteinase inhibitors, the rSPIPm5 could inhibit the hemolymph proPO activity. In survival experiments, the rSPIPm5 could prolong the life of WSSV-infected shrimp similar to the effect of heat stress. The rSPIPm5 also helped the YHV-, Vibrio harveyi- and V. parahaemolyticus-infected shrimp survive longer. The increased endurance against microbial infection was due to the inhibitory effects presumably activated by rSPIPm5 on viral replication and bacterial growth but not the expression of antimicrobial peptides. Therefore, the SPIPm5 plays an important role in shrimp innate immunity against the viral and bacterial infection.