Two sgRNAs were designed to target the region of exon 2 of the pULBP1 gene by microinjection. The co-injection of modified Cas9-D10A nickase with a pair of sgRNAs into the zygote's cytoplasm easily and efficiently generated biallelic modification of the pULBP1 gene in one step. Five out of nine F0 generation piglets showed insertions or deletions in the targeting site of the pULBP1 gene, indicating that pULBP1 mutation efficiency reached about 56% (5/9). Quantitative determination of pULBP1 showed approximately a 1.53-fold reduction in the amount of protein ULBP1 on the cell surface (ELISA). A human NK-cell cytotoxicity test leads to the conclusion that higher cell viability is observed for -/- ULBP1 (survival rate 85.36%) compared to +/+ ULBP1 (69.58%). ULBP1-KO pigs will provide a more progressive xenograft source for further research studies, especially those measuring the effects of abolishing the gene function in terms of the complexity of the immunological interactions.