The SECIS element is an RNA hairpin in the 3′UTR of selenoprotein mRNAs required for decoding UGA selenocysteine codons. Our experimentally derived 2D structure model for the SECIS RNA revealed the conservation of four consecutive non-Watson–Crick base pairs, with a central G.A/A.G tandem. The present study was dedicated to gaining insight into the role of this quartet of base pairs. The effects of mutations introduced into the SECIS quartet of the glutathione peroxidase (GPx) cDNA, an enzyme with selenocysteine in its active center, were reported in vivo by the GPx activity. The detrimental consequence of an all-Watson–Crick mutant quartet disclosed the paramount importance of the non-Watson–Crick base pairs for GPx activity. Next, structure probing established that base pair changes in the central G.A/A.G tandem, predicted by the model to be structurally unfavorable, effectively led to local opening of the helix at the quartet. A concomitant abolition of GPx activity was observed, arising from translational impairment of full-length GPx. In contrast, an isosteric base pair replacement in the tandem did not affect base pairing in the quartet, leading to an almost wt GPx activity. Collectively, the data provided conclusive evidence for the functional relevance of these non-Watson–Crick base pairs in vivo, thus identifying a noncanonical RNA motif crucial to SECIS function in mediating selenoprotein translation. Within the quartet, the prominent requirement for the central G.A/A.G tandem is highlighted, our previous structural model and the mutagenesis data presented here strongly arguing in favor of a sheared arrangement for the G.A base pairs. The SECIS RNA is therefore another member to be added to the growing list of RNAs containing building blocks of non-Watson–Crick base pairs, required for structure and/or function.