Porcine epidemic diarrhea (PED) to date remains a colossal burden to the global swine industries owing to the lack of successful vaccine in the field [12,13]. Moreover, other emerging and re-emerging swine enteric coronaviruses, including porcine deltacoronavirus (PDCoV) [24], and swine acute diarrhea syndrome coronavirus (SADS-CoV) [25], further complicate the field condition by affecting diagnostic accuracy and increasing the risk of viral recombination [26]. Hence, there is still a pressing need to develop a safe and effective vaccine, particularly the LAV, to mount this disastrous disease. The reverse genetics system is a powerful and widely used tool to study viral pathogenesis and novel LAV design by active modification of genes of interests. Previous studies using reverse genetics on other coronaviruses have identified many virulent/attenuating determinants that might be shared among different genus of coronaviruses [12]. Nevertheless, direct evidences of attenuation due to harboring those mutated determinant(s) in pigs are still limited in PEDV [16,23,27]. In the present study, to investigate the role of the S gene in the attenuation process of the PEDVPT 52 strain, we generated four infectious cDNA clones of G2b PEDV, including the parental virulent iPEDVPT-P5, attenuated iPEDVPT-P96, as well as two chimeric viruses (iPEDVPT-P5-96S and iPEDVPT-P96-5S) with exchanged S gene. We found that the iPEDVPT-P96 virus fully regained the virulence after the exchange of S gene derived from the highly virulent iPEDVPT-P5 virus, showing comparable patterns of viral shedding, diarrhea and histopathological changes. Our data confirmed that the S gene is the primary attenuating determinant of the iPEDVPT-P96 virus and the genetic backbone other than the S gene involving the attenuation of the iPEDVPT-P96 might be of less importance. However, the iPEDVPT-P5-96S still exhibited partial virulence resulting in severer villous attenuation and higher mortality rate compared to those caused by the iPEDVPT-P96 virus. This result supports that the virulence of PEDV might be a multigenic event.