To construct of an eukaryotic expression vector encoding herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2), an Iranian isolate of HSV-2 was propagated in HeLa cell line and its DNA was extracted and used as template in polymerase chain reactions (PCR), to amplify gD2 gene. Primers were designed and the restriction enzyme sites for EcoRI and XhoI were considered at their 5′ ends respectively. The PCR product was confirmed by restriction enzyme analysis, cloned into a cloning vector (pBsc) and then sequenced. The fragment encoding gD2 was obtained by digestion using appropriate enzymes and extracted on agarose gel using a commercial kit. The gene of interest was subcloned in the named sites of an eukaryotic expression vector (pcDNA3) to construct pcDNA-gD2. An endotoxin free column was applied to prepare pure pcDNA-gD2, which was transfected into mammalian cells using lipofectamine. Protein expression was confirmed using indirect immunofloerscent test. The results indicated that the construct expresses in mammalian cell lines effectively and it can be used as a DNA vaccine in animal models.