It is well-accepted that gene expression is heavily influenced by RNA structure. For instance, stem-loops and G-quadruplexes (rG4s) are dynamic motifs in mRNAs that influence gene expression. Adenosine-to-inosine (A-to-I) editing is a common chemical modification of RNA which introduces a nucleobase that is iso-structural with guanine, thereby changing RNA base-pairing properties. Here, we provide biophysical, chemical, and biological evidence that A-to-I exchange can activate latent rG4s by filling incomplete G-quartets with inosine. We demonstrate the formation of inosine-containing rG4s (GI-quadruplexes) in vitro and verify their activity in cells. GI-quadruplexes adopt parallel topologies, stabilized by potassium ions. They exhibit moderately reduced thermal stability compared to conventional G-quadruplexes. To study inosine-induced structural changes in a naturally occurring RNA, we use a synthetic approach that enables site-specific inosine incorporation in long RNAs. In summary, RNA GI-quadruplexes are a previously unrecognized structural motif that may contribute to the regulation of gene expression in vivo.