MicroRNA (miRNA), a class of non-coding RNA (about 22 nt), widely exists in different organisms and plays an important role in regulating cell growth, development, and apoptosis. Recently, various studies have demonstrated that miRNA also serves as a key mediator during testis development and spermatogenesis both in human beings and animals. However, the expression of miRNA in different testis from different species showed significant differences. Meanwhile, it also showed different expression patterns in different spermatogenesis stages. In this review, we summarize different miRNA expression patterns during testis development, spermatogenesis, as well as their roles in regulating spermatogenesis, which provids insight into the further research on miRNA in testis, and ideas for regulating and improving animal sperm quality by using miRNA.