In this study, the first pair of cDNA clones for a virulent G2b PEDV, and its derived attenuated strain were generated, allowing us to use both gain-of-function and lose-of-function approaches to studying the role of the S gene in PEDV pathogenesis. We confirmed that the S gene is a crucial virulent/attenuating determinant for the iPEDVPT-P96, but its importance varies among different PEDV strains. Thus, other studies’ results alongside our results will provide valuable information for the future generation of novel chimeric or multivalent vaccines.