Since co-suppression was discovered as a pioneer silencing phenomenon of RNA interference (RNAi) in petunia in 1990, many types of small RNAs have been identified in the RNAi pathway among various eukaryotes. In plants, a large number of 21- or 24-nucleotide (nt) phased small interfering RNAs (phasiRNAs) are produced via processing of long RNA precursors by Dicer-like proteins. However, the roles of phasiRNAs remain largely unknown. The development of imaging technology and RNA profiling has clarified the spatiotemporal regulation of phasiRNAs, and subsequently the different functions of 21-nt trans-acting phasiRNAs and 24-nt cis-regulatory phasiRNAs during male organ development. This review focuses on the biogenesis, diversification, spatiotemporal expression pattern and function of phasiRNAs in plants.