Piwi-interacting RNAs (piRNAs) are a distinct class of small non-coding RNAs specifically expressed in the germline of many species. Studies show that the piRNA pathway influences spermatogenesis by translation regulation, germline stem cell maintenance, RNA degradation and gene defense in addition to inhibition of transposable elements. This review presents an overview of the piRNA pathway focusing on piRNA, PIWI protein and other related proteins and outlines the latest advances in the studies of the piRNA pathway in spermatogenesis.