Hepatocyte nuclear factor 4-alpha (HNF4α) is a well-established master regulator of liver development and function. Restoration of HNF4α can treat multiple liver disorders and liver cancers. To date， HNF4α is still "undruggable" due to lack of known activating ligands. Thus， understanding the regulatory mechanism of HNF4α expression may help develop an alternative approach to modulate HNF4α protein levels. G-quadruplexes (G4) are non-canonical stable secondary structures discovered mostly in the promoters of oncogenes. Recent genome-wide studies demonstrate the enrichment of G4s in the 5' untranslated region (UTR). By protoporphyrin IX-binding assay and circular dichroism spectrum， we validated the presence of a chemically highly stable 4-ring G4 within the 5' UTR of mouse Hnf4a1. Our real-time PCR and Western blot data showed that the Hnf4a1 5' UTR caused a remarkable translational suppression regardless of a moderate effect on Hnf4a1 mRNA levels. The subsequent deletion/mutation analysis of Hnf4a1 5' UTR using dual-luciferase reporter assays further demonstrated that although the disruption of the chemically highly stable 4-ring G4 resulted in a marked attenuation of inhibition， the G4 alone only weakly inhibited translation. Likewise， disruption of a long stem-loop adjacent to the 4-ring G4 markedly attenuated translational inhibition， although the stem-loop alone only exerted a weak inhibitory effect. Thus， the tight conjunction of G4s and an adjacent stem-loop within the Hnf4a1 5' UTR was both necessary and sufficient to mediate the very strong translational repression. Our results establish a novel working model that a chemically stable G4 may require co-factors to be bio-stable for exerting biological functions.