In our previous work, prohibitin1 (PHB1) was identified to be only expressed in granulocytes of Fenneropenaeus chinensis. In order to elucidate the potential immunological properties of prohibitins in hemocyte subpopulations, in this paper, the full-length cDNAs of PHB1 and PHB2 were firstly cloned from F. chinensis using rapid amplification of cDNA ends approach, and they were designated FcPHB1 and FcPHB2, respectively. Based on the sequence analysis and multiple sequence alignment, FcPHB1 and FcPHB2 were members of SPFH protein family. By quantitative real-time RT-PCR, the higher mRNA transcription levels of FcPHB1 and FcPHB2 were detected in intestine and hemocytes of F. chinensis, and these two genes in hemocytes were significantly up-regulated upon WSSV infection. The FcPHB1 and FcPHB2 were recombinantly expressed in Escherichia coli BL21 (DE3), and employed as immunogens to produce the polyclonal antibodies (PAbs) in rabbits. Indirect immunofluorescence assay (IFA) revealed that the FcPHB1 and FcPHB2 were located both in the cytoplasm and nuclei of hemocytes, which could also be specifically recognized by the PAbs against FcPHB1 or FcPHB2 in Western blot. Interestingly, it was found that FcPHB1 and FcPHB2 were only expressed in the granulocytes of heathy shrimp and highly expressed in the WSSV-infected granulocytes, however only weak expressions of FcPHB1 and FcPHB2 were observed in the hyalinocytes of WSSV-infected shrimp. Meanwhile, silencing of FcPHB1 and FcPHB2 genes were performed by small interfering RNA, and the results showed that the WSSV copies in hemocytes were increased by knockdown of either FcPHB1 or FcPHB2, and the cumulative mortalities of shrimp in the silenced groups were also markedly increased. These results demonstrated that FcPHB1 and FcPHB2 played important roles in anti-WSSV infection, and their differential expression characteristics in hemocyte subpopulations provided a further understanding of the immune functions of granulocytes and hyalinocytes.