RNA-mediated gene regulation and expression are critically dependent on both nucleic acid architecture and recognition. We present a novel mechanism for the regulation of gene expression through direct RNA-RNA interactions between small RNA and mRNA in human cells. Using mRNA reporters containing G-rich sequences in the 5'-untranslated region (5'-UTR), in the coding region, or both, we showed that G-rich small RNAs bind to the reporter mRNAs and form an intermolecular RNA G-quadruplex that can inhibit gene translation in living cells. Using a combination of circular dichroism (CD) and RNase footprinting in vitro, we found that the intermolecular G-quadruplexes show a parallel G-quadruplex structure. We next investigated whether the intermolecular G-quadruplex is present in living cells. Employing the fluorophore-labeled probes, we found that two G-rich RNA molecules form an intermolecular G-quadruplex structure in living cells. These results extend the concept of small RNA-mediated expression and suggest an important role for such RNA structures in the inhibition of mRNA translation.