Abstract: Porcine reproductive and respiratory syndrome (PRRS) is a worldwide contagious disease caused by PRRSV, a positive-strand RNA virus, resulting in significant economic losses in the swine industry. Deleting or substituting the CD163 SRCR5 domain can effectively resist PRRSV infection in pigs. However, large fragment modifications in pigs inevitably raise concerns about their growth performance. Reducing the impact of gene modifications on normal physiological functions is a promising direction for breeding PRRSV-resistant pigs. Here, we identified a functional amino acid in CD163 that affects PRRSV proliferation. Virus infection experiments on Marc145 and PK-15CD163 cells illustrated that the mE535G or corresponding pE529G mutation significantly inhibited HP-PRRSV proliferation via preventing PRRSV binding and entry. Furthermore, individual viral challenge tests revealed that E529G pigs had viral loads two orders of magnitude lower than wild type (WT) pigs, confirming that E529G pigs could effectively resist HP-PRRSV. Examination of physiological indicators and the scavenger function of CD163 in pigs verified no difference between WT and E529G pigs. This highlights that E529G pigs could be used for breeding PRRSV-resistant pigs and provides new insights into controlling future PRRSV outbreaks.