Hyphantria cunea nucleopolyhedrovirus (HycuNPV) infection protected SpIm cells from actinomycin D (ActD)-induced apoptosis as early as 4 h postinfection. Analysis by Southern hybridization revealed that the HycuNPV genome possessed three members of inhibitor of apoptosis genes (iaps) that were designated as hycu-iap1, hycu-iap2, and hycu-iap3 because of their amino acid sequence homology with iaps identified in other baculoviruses. Functional analysis of Hycu-IAPs by transient expression assay in Sf9 cells revealed that Hycu-IAP3 blocked apoptosis induced by actinomycin D and rescued replication of p35 deficient-mutant AcMNPV, while Hycu-IAP1 and Hycu-IAP2 did not show any anti-apoptotic functions. Knockdown of hycu-iap3 expression by RNAi during HycuNPV infection in SpIm cells induced apoptosis. These results indicate that Hycu-IAP3 is essential for blockage of apoptosis during HycuNPV infection of permissive SpIm cells.