The oncogene c-MYC has guanine-rich and complementary cytosine-rich sequences in its P1 promoter region. The P1 promoter is responsible for over 90% of the c-MYC expression. Downregulation of c-MYC expression represents a novel therapeutic approach to more than 50% of all cancers. A stable i-motif formed by the c-MYC C-rich sequence would be an attractive target for cancer treatment. We have previously shown that c-MYC promoter sequences can form stable i-motifs in acidic solution (pH 4.5-5.5). The question is whether c-MYC promoter sequence i-motif will be stable at physiological pH. In this work, we have investigated the stability of mutant c-MYC i-motif in solutions having pH values from 4 to 7 and containing co-solutes or molecular crowding agents. The crowded nuclear environment was modeled by the addition of polyethylene glycol (PEG, having molecular weights from 200 to 12000 g/mol) at concentrations of 10% to 40% w/w. Circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC) were used to establish the presence and stability of c-MYC i-motifs in buffer solutions having pH values of 4 to 7. The results of these studies are: 1) the addition of up to 20% w/w glycerol does not increase i-motif stability, 2) the addition of 30% PEG results in an increase in i-motif stability to pH values as high as 6.7, 3) i-motif stability is increased with increased PEG concentration and increased PEG molecular weight, and 4) the effects of PEG size and concentration are not linear, with larger PEGs forming DNA/PEG complexes, which destabilize the i-motif. In summary, we have shown that the c-MYC i-motif can exist as a stable structure at pH as high as 6.7 in a crowded environment. Molecular crowding, largely an excluded volume effect, drives the formation of the more compact i-motif, even at higher pH values where the cytosine imino-nitrogen is deprotonated and neutral C-C pairs can form only two H-bonds. Based on this research, it seems possible that a stable c-MYC promoter sequence i-motif could form at physiological pH and would be a reasonable drug target for new cancer therapies.