As the most abundant small RNAs, piwi-interacting RNAs (piRNAs) have been identified as a new class of non-coding RNAs with 24-32 nucleotides in length, and they are expressed at high levels in male germ cells. PiRNAs have been implicated in the regulation of several biological processes, including cell differentiation, development, and male reproduction. In this review, we focused on the functions and molecular mechanisms of piRNAs in controlling spermatogenesis, including genome stability, regulation of gene expression, and male germ cell development. The piRNA pathways include two major pathways, namely the pre-pachytene piRNA pathway and the pachytene piRNA pathway. In the pre-pachytene stage, piRNAs are involved in chromosome remodeling and gene expression regulation to maintain genome stability by inhibiting transposon activity. In the pachytene stage, piRNAs mediate the development of male germ cells via regulating gene expression by binding to mRNA and RNA cleavage. We further discussed the correlations between the abnormalities of piRNAs and male infertility and the prospective of piRNAs' applications in reproductive medicine and future studies. This review provides novel insights into mechanisms underlying mammalian spermatogenesis and offers new targets for diagnosing and treating male infertility.