Aim: Surface-displayed heterologous antigens on Bacillus subtilis spores can induce the vertebrate animals tested to generate local and systematic immune response through oral immunization. Here, the protection potential of the recombinant spores displaying the VP28 protein of white spot syndrome virus (WSSV) was investigated in the invertebrate crayfish (Cambarus clarkii). Methods and Results: The VP28 protein was successfully displayed on the surfaces of B. subtilis spores using CotB or CotC as a fusion partner. Crayfish were administrated orally by feeding the feed pellets coated with B. subtilis spores for 7 days and immediately followed by WSSV challenge. Oral administration of either spores expressing CotB-VP28 or CotC-VP28 resulted in significantly higher relative survival rates of 37.9 and 44.8% compared with the crayfish orally administrated with the spores nonexpressing VP28 (10.3% relative survival rate). When challenges were separately conducted at 7 and 21 days after oral administration, the relative survival rates increased to 46 4 and 50% at 7 days post-oral administration, but decreased to 30 and 33.3% at 21 days after oral administration. Conclusion: These evidences indicate that the surface-displayed VP28 on B. subtilis spore could induce protection of crayfish against WSSV via oral administration. Significance and Impact of the Study: This is the first report to use the spore surface display system to deliver orally a heterologous antigen in an aquatic invertebrate animal, crayfish. The results presented here suggest that the spore-displayed VP28 might be suitable for an oral booster vaccine on prevention of WSSV infection in shrimp farming.