Change in cellular glucose metabolism is considered to be a biochemical hallmark in cancer cells. The mitochondrion is the key organelle in which glucose metabolism occurs. However, whether DNA methylation at the displacement loop (D-loop) region of mitochondrial DNA (mtDNA) has an effect on the expression of the rate-limiting enzyme, and, therefore, on oxidative phosphorylation in colorectal cancer remains to be determined. Quantitative change in ND2 (a subunit of NADH) and the methylation status of the D-loop were observed during the initiation and progression of colorectal cancer. Furthermore, the possible correlations with clinicopathological stage were also investigated. Tumor and corresponding non-cancerous tissues were surgically resected from 44 colorectal cancer patients between 2008 and 2009. Cox IV expression was quantified in all of the specimens, and the ND2 expression was calculated. Quantitative changes in ND2 expression exhibited a significant increase. The average relative ratios of ND2 content were 1.67±0.44 in the tumor tissues and 0.89±0.44 in the corresponding non-cancerous tissues (p<0.01). In addition, the D-loop of most corresponding non-cancerous tissues was methylated and the percentage was 79.5%, while this percentage was much smaller in the tumor tissues (11.4%). Following correlation with clinicopathological data, changes in the ND2 expression in the colorectal cancer exhibited a significant association with clinicopathological stage. This increase was significant as early as in stage Ⅰ. Furthermore, the ratios of unmethylated D-loop cases were increased in both tumor and corresponding non-cancerous tissues, and the ND2 expression was also increased from stages Ⅰ to Ⅳ. Our results indicate that demethylation of the D-loop plays a key role in regulating ND2 expression during the initiation and/or progression of colorectal cancer.