To analyse the function of the duck plague virus (DPV) glycoprotein J homologue (gJ), two different mutated viruses, a gJ deleted mutant ΔgJ and a gJR rescue mutant gJR with US5 restored were generated. All recombinant viruses were constructed by using two-step of RED recombination system implemented on the duck plague virus Chinese virulent strain (DPV CHv) genome cloned into a bacterial artificial chromosome. DPV-mutants were characterized on non-complementing DEF cells compared with parental virus. Viral replication kinetics of intracellular and extracellular viruses revealed that the ΔgJ virus produce a 10-fold reduction of viral titers than the gJR and parental virus, which especially the production of extracellular infectivity was affected. In addition, the ΔgJ virus produced viral plaques on DEF cells that was on average approximately 11% smaller than those produced by the gJR and parental viruses. Electron microscopy confirmed that although DPV CHv without gJ could efficiently carry out viral replication, virion assembly and envelopment within infected cells, the ΔgJ virus produced and accumulated high levels of anuclear particles in the nuclear and cytoplasm. These results show that the gJ slightly impaired in viral replication, virion assembly and cell-to-cell spread, and is not essential in virion envelopment.