Aptamers bind to their targets with exceptional affinity and specificity. However, their intracellular application is hampered by the lack of knowledge about the effect of the cellular milieu on the RNA structure/stability. In this study, cellular crowding was mimicked using polyethylene glycol (PEG), and the crucial role of Mg2+ ions in stabilizing the structure of an RNA aptamer was investigated. Increasing the concentration of Mg2+ or PEG increased the thermal stability of the aptamer. The crowding effect lowered the requirement of the Mg2+ ion to form the binding-competent conformer of the aptamer. This suggests that crowding and other factors may compensate for a lower concentration of Mg2+ for proper folding of aptamers inside cells. Selective 2'-hydroxyl acylation and primer extension (SHAPE) probing permitted residue-level analysis of the aptamer. Mg2+ and/or PEG were shown to be involved in increasing the rigidity or flexibility of different regions of the aptamer. Fluorescence correlation spectroscopy showed a significantly low hydrodynamic radius (RH) in the presence of molecular crowders and Mg2+ ions. We believe that the decreased water activity due to crowding may be responsible for reduced RH. Our results show that in a crowded environment, the RNA aptamer was exposed to conformers that were not available to it in simple buffer solutions or solely in the presence of lower concentrations of Mg2+.