Background & Aims: RNA G-quadruplexes (RG4s) appear to be important in post-transcriptional gene regulation, but their pathophysiological functions remain unknown. MicroRNA-26a (miR-26a) is emerging as a therapeutic target for various human diseases, however the mechanisms underlying endogenous miR-26a regulation are poorly understood. Herein, we study the role of RG4 in miR-26a expression and function in vitro and in vivo. Methods: Putative RG4s within liver-enriched miRNAs were predicted by bioinformatic analysis, and the presence of an RG4 structure in the miR-26a-1 precursor (pre-miR-26a-1) was further analyzed by biophysical and biochemical methods. RG4 stabilizers, pre-miR-26a-1 overexpression plasmids, and luciferase reporter assays were used to assess the effect of RG4 on pre-miR-26a-1 maturation. Both miR-26a knock-in and knockout mouse models were employed to investigate the influence of this RG4 on miR-26a expression and function. Moreover, the interaction between RG4 in pre-miR-26a-1 and DEAH-box helicase 36 (DHX36) was determined by biophysical and molecular methods. Finally, miR-26a processing and DHX36 expression were quantified in the livers of obese mice. Results: We identify a guanine-rich sequence in pre-miR-26a-1 that can fold into an RG4 structure. This RG4 impairs pre-miR-26a-1 maturation, resulting in a decrease in miR-26a expression and subsequently an increase in miR-26a cognate targets. In line with known miR-26a functions, this RG4 can regulate hepatic insulin sensitivity and lipid metabolism in vitro and in vivo. Furthermore, we reveal that DHX36 can bind and unwind this RG4 structure, thereby enhancing miR-26a maturation. Intriguingly, there is a concordant decrease of miR-26a maturation and DHX36 expression in obese mouse livers. Conclusions: Our findings define a dynamic DHX36/RG4/miR-26a regulatory axis during obesity, highlighting an important role of RG4 in physiology and pathology. Lay summary: Specific RNA sequences called G-quadruplexes (or RG4) appear to be important in post-transcriptional gene regulation. Obesity leads to the formation of these RG4 structures in pre-miR-26a-1 molecules, impairing the maturation and function of miR-26a, which has emerged as a therapeutic target in several diseases. This contributes to hepatic insulin resistance and the dysregulation of liver metabolism. (c) 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.