Seasonal reproduction is a mammalian behavior that has developed over an extended evolutionary period and requires animals to respond to external environmental changes to facilitate reproduction. In this study, we investigated the role of PIWI-interacting RNA (piRNA) in the seasonal reproduction of plateau zokors (Eospalax baileyi). piRNA expression profiles in plateau zokor testes during both breeding and non-breeding seasons were examined. The piRNAs had a distinctive ping-pong signature and ranged from 27 to 32 nt with a peak at 30 nt. Testicular piRNAs predominantly aligned to specific genomic regions, including repeat and gene regions. Analysis of the piRNA–mRNA interaction network and functional enrichment of differentially expressed piRNAs targeting mRNAs revealed their association with testicular development and spermatogenesis. Significantly, PIWIL4 is an mRNA gene that interacts with piRNA and exhibits high expression levels within the testes during the non-breeding phase. This study provides a foundation to improve our understanding of piRNA regulatory mechanisms during testicular development and spermatogenesis in seasonally reproducing animals and, specifically, in the plateau zokor.