The global practice of cryopreservation of human semen is commonplace in Assisted Reproductive Technology (ART) labs and sperm banks. However, information on the effects of long-term cryopreservation on semen is limited to clinical data summaries and descriptions. For this study, we prepared 4 semen specimens of fresh semen, 4 specimens cryostored for at least 1 year, 3 specimens cryostored for at least 5 years, 4 specimens cryostored for at least 10 years, and 3 specimens cryostored for at least 15 years. Total RNA was extracted from each sample, amplified, labeled, and mapped to the known primary microRNA (miRNA) in the miRBase database, enabling the prediction of novel miRNAs. We found that cryopreservation can lead to changes in miRNA expression, and with the increase in storage time, these changes became more pronounced. Meanwhile, the expression of let-7d-3p, let-7c-5p and let-7i-3p miRNAs changed dynamically over cryostorage time in frozen-thawed human sperm. Furthermore, we analyzed the time-dependent dynamics of cryostorage-expressed miRNAs and their target mRNAs and found that half of the target genes were expressed in oocytes. These intersection genes were mainly enriched in cancer and cytoskeletal signaling pathways. Our findings showed that the miRNA expression profile of cryopreserved human semen is modified by long-term storage. Furthermore, as the storage time increases, the impact on human sperm becomes more pronounced in terms of miRNAs, which may have an effect on subsequent fertilization and embryonic development.