Wheat stripe rust, caused by the obligate biotrophic fungusPuccinia striiformisf. sp.tritici(Pst), seriously affects wheat production. Here, we report the complete genome sequence and biological characterization of a new mitovirus fromP. striiformisstrain GS-1, which was designated as “Puccinia striiformis mitovirus 2” (PsMV2). Genome sequence analysis showed that PsMV2 is 2658 nt in length with an AU-rich of 52.3% and comprises a single ORF of 2348 nt encoding an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis indicated that PsMV2 is a new member of the genusUnuamitoviruswithin the familyMitoviridae.In addition, PsMV2 multiplied highly duringPstinfection and it suppresses programmed cell death (PCD) triggered by Bax. Silencing of PsMV2 inPstby barley stripe mosaic virus (BSMV)-mediated Host Induced Gene Silencing (HIGS) reduced fungal growth and decreased pathogenicity ofPst. These results indicate PsMV2 promotes host pathogenicity inPst. Interestingly, PsMV2 was detected among a wide range of field isolates ofPstand may have coevolved withPstin earlier times. Taken together, our results characterized a novel mitovirus PsMV2 in wheat stripe rust fungus, which promotes the virulence of its fungal host and wide distribution inPstwhich may offer new strategies for disease control.