Objective Seminal plasma exosomes (spEXs) play a crucial role in sperm maturation, apoptosis and fertilization. This study was aimed to explore the miRNA expression of boar spEXs and the potential regulatory role of miRNA in sperm maturation and functional maintenance.Method The spEXs from Yorkshire boar semen were isolated and the exosomes were identified through transmission electron microscopy, nanoparticle tracking analysis, marker protein expression analysis and high-throughput miRNA sequencing. Result The spEXs were successfully isolated. A total of 329 spEX miRNAs were identified by miRNA sequencing. Through target gene prediction and functional enrichment analysis of highly expressed miRNAs, it was concluded that spEX miRNAs played potential regulatory roles in sperm ejaculation, P53 signaling pathway, prostate cancer, cell response to DNA damage stimuli, negative regulation of apoptosis, acrosome membrane binding, fertilization, etc. Conclusion This study provides basic data for spEX miRNAs regulating sperm motility and sperm fertilization, and provides references for studying the regulation mechanism of semen preservation.