The polyhedrin is responsible to form polyhedra of nucleopolyhedrovirus(NPV) and highly conserved in most completely sequenced in lepidopteran NPVs. Previously, we have reported that the substitution of polyhedrin of Autographa californica NPV(AcNPV) with that of Spodoptera exigua NPV(SeNPV) or Bombyx mori NPV(BmNPV) result the change of polyhedra morphology. In this study, we investigated the influence of changed polyhedra morphology to the virulence of AcNPV. The recombinant AcNPVs were propagated in Spodoptera frugiperda clone 9, 21 cells and S. exigua larvae. Each collected recombinant polyhedra were used in bioassays using S. exigua larvae. The recombinant AcNPVs show that difference virulence according to the polyhedra morphologies. Internal and external morphological features of each recombinant AcNPV were also compared on the electron microscope. Our results suggest that the morphology of polyhedra influence the virulence of NPV and is well worth considering for the development viral insecticide.