The present study generated nectin1-mutant mice with single amino acid substitution and tested the anti-pseudorabies virus (PRV) ability of the mutant mice, with the aim to establish a model for PRV-resistant livestock. A phenylalanine to alanine transition at position 129 (F129A) of nectin1 was introduced into the mouse genome to generate nectin1 (F129A) mutant mice. The mutant mice were infected with a field-isolated highly virulent PRV strain by subcutaneous injection of virus. We found that the homozygous mutant mice had significantly alleviated disease manifestations and decreased death rate and viral loading in serum and tissue compared with heterozygous mutant and wild-type mice. In addition to disease resistance, the homozygous mutant mice showed a defect in eye development, indicating the side effect on animals by only one amino acid substitution in nectin1. Results demonstrate that gene modification in nectin1 is an effective approach to confer PRV resistance on animals, but the mutagenesis pattern requires further investigation to increase viral resistance without negative effect on animal development.