In the present study, we utilized reverse genetics to examine the role of the S gene in the attenuation process of PEDV using the highly virulent and attenuated G2b PEDV Pintung (PEDVPT) 52 strains, the PEDVPT-P5 and PEDVPT-P96 viruses. Nonetheless, our findings were discordant with previous reported data [17]. We found that replacement of the S gene with the iPEDVPT-P5 virus enabled iPEDVPT-P96 to regain its virulence. A reciprocal approach revealed that iPEDVPT-P5 virus became partly attenuated after the S gene was exchanged to iPEDVPT-P96. Collectively, we concluded that the S gene is of critical importance to the attenuation process of the PEDVPT 52 strain. However, mutation in the S gene alone could not completely attenuate the iPEDVPT-P5 virus. Thus, gene(s) other than S gene could also play a role in determining the virulence.