Nuclease 1 (NUC1) is a highly conserved nonspecific endonuclease present in vertebrates, fungi, and a few plant species. While the involvement of yeast NUC1p in apoptotic cell death independent of metacaspase or apoptosis-inducing factors is documented, its function in other fungi, particularly pathogenic ones, remains elusive. In this study, we identified and characterised the homologue of yeast NUC1p, termed PstNUC1, in Puccinia striiformis f. sp. tritici (Pst), the causal agent of wheat stripe rust. PstNUC1 was induced during infection and was secreted to the extracellular space of the host. Silencing of PstNUC1 significantly attenuated the virulence of Pst, suggesting its critical role in pathogenicity. Exogenous treatment of PstNUC1 diminished the extracellular DNA (exDNA)-triggered plant immune response, including cell death, oxygen peroxide production, and upregulation of Pathogenesis-related genes. Notably, overexpression of wild-type PstNUC1, but not a signal peptide-deficient mutant (PstNUC1