Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe economic losses to current swine production worldwide. Highly pathogenic PRRSV (HP-PRRSV), originated from a genotype 2 PRRSV, is more virulent than classical PRRSV and further exacerbates the economic impact. HP-PRRSV has become the predominant circulating field strain in China since 2006. CD163 is a cellular receptor for PRRSV. The depletion of CD163 whole protein or SRCR5 region (interaction site for the virus) confers resistance to infection of several PRRSV isolates in pigs or cultured host cells. In this study, we described the generation of a CD163 knockout (KO) pig in which the CD163 protein was ablated by using CRISPR/Cas9 gene targeting and somatic cell nuclear transfer (SCNT) technologies. Challenge with HP-PRRSV TP strain showed that CD163 KO pigs are completely resistant to viral infection manifested by the absence of viremia, antibody response, high fever or any other PRRS-associated clinical signs. By comparison, wild-type (WT) controls displayed typical signs of PRRSV infection and died within 2 weeks after infection. Deletion of CD163 showed no adverse effects to the macrophages on immunophenotyping and biological activity as hemoglobin–haptoglobin scavenger. The results demonstrated that CD163 knockout confers full resistance to HP-PRRSV infection to pigs without impairing the biological function associated with the gene.