The two important epigenetic markers in the human genome, 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC), are involved in gene regulation processes. As a major epigenetic target, cytosines in a C-rich DNA sequence were substituted with mC and hmC to investigate the thermal stability and pH sensitivity of the corresponding i-motifs. Circular Dichroism (CD) studies indicate the formation of i-motifs at acidic pH (<6.5) for mC- and hmC-modified DNA sequences. Thermal denaturation results suggest that DNA i-motifs are stabilized when modified with one or two mCs. However, hypermethylation with mC and single modification with hmC cause destabilization of the structure. A biomimetic crowding agent does not alter the stability effect trends resulting from mC and hmC modifications, though the corresponding i-motifs show elevated melting temperatures without significant changes in pKa values.