Here we identify hundreds of RNA G-quadruplex (rG4) candidates in microRNAs (miRNAs), characterize the miRNA structure and miRNA-mRNA interactions on several mammalian-conserved miRNAs, and reveal the formation of rG4s in miRNAs. Notably, we study the effect of these rG4s in cells and uncover the role of rG4s in miRNA-mediated post-transcriptional regulation.