Objective: Peroxisomal Protein (PEP) encodes one of the proxisomal matrix proteins, termed PeP, consists 209 amino acid resdiues. PeP contains a three peptide, SKL, at its C-terminus, acts as protein targeting signal (PTS1). Recent study on PeP expression, which was carried out in Royan Institute, demonstrated that PEP expression level elevated during the process of neurogenesis process. In order to clarify the PeP function in neural differentiation of mouse embryonic stem cells the present study was carried out. Materials and Methods: several siRNAs to silence PeP gene expression were designated using online softwares for siRNA designing (Ambion, Dharmacon, Invitrogen, SiDirect, Genescript). The suggested most appropriated siRNAs were 19nt in length along with a negative control siRNA (scramble) These chosen siRNAs were checked for secondary structure formation using Genebee and mfold softwares. At last siRNAs changed to shRNAs with inserting a sequence loop between the sense and anti sense sequences and were ordered to synthesis. Synthesized oligonucleotides were annealed and inserted into the vector at HindIII and XhoI sites. The recombinant vectors were checked by digestion with MluI and sequencing. The recombinant vectors were transected in to the mouse embryonic stem cells. Stably transformed colony cells expressing PEP shRNA were selected after antibiotics treatment of the transfected cells. Doxycycline was used to induce the production of PEP shRNA and silencing of PEP gene in mouse embryonic stem cells and during EBs formation . Real time quatitative RT-PCR revealed that one of the constructed PEP shRNA had a significant decrease in PEP expression. Results: Among three different shRNA structures, 2644 and 499 shRNA structures knocked down PeP gene expression in mouse embryonic stem cells after 48 hours doxycyclin treatment. 2644 shRNA structure decreased PeP expression about 78% relative to scrambel (negative control)also this expression reduction was observed in Retinoic acid- treated EBs in compare to scramble. Conclusion: Knock down of Pep expression was observed significantly in mouse embryonic stem cells and EBs treated by retinoic acid .these results show that 2644 structure more effective than others for knock down of target gene.