Mitochondrial DNA (mtDNA) methylation may be associated with mitochondrial damage; this study investigates their relationship in contrast-induced renal tubular epithelial cell (RTEC) injury. We stimulated HK-2 cells with iohexol to establish an model and analyzed the methylation level of mtDNA by bisulfite amplicon sequencing. The mitochondrial membrane potential, mitochondrial reactive oxygen species (mtROS), intracellular ROS, and changes in mitochondrial ultrastructure were evaluated as indicators of mitochondrial damage. Iohexol significantly inhibited cell viability and induced cell apoptosis, increasing both mtROS and intracellular ROS levels. Additionally, the methylation levels of mtDNA-encoded genes cytochrome c oxidase subunit I (COX I) (3.09%, * < 0.05), cytochrome c oxidase subunit II (COX II) (4.51%, ** < 0.01), cytochrome c oxidase subunit III (COX III) (3.50%, ** < 0.01) and cytochrome B (CYTB)(4.66%, * < 0.05) were increased, accompanied by enhanced transcription of both COX I and COX III. 5-Aza-dC, as a DNA methylation inhibitor, was dissolved in dimethyl sulfoxide (DMSO) vehicle to explore the role and mechanism of inhibiting mtDNA methylation in contrast-induced RTEC injury. HK-2 cells were further divided into four groups: vehicle control (DMSO alone), vehicle pretreated contrast - induced group (CI) (DMSO-CI), inhibitor control (5-Aza-dC), and inhibitor pretreated CI (5-Aza-dC-CI). Intriguingly, administration of 5-Aza-dC effectively attenuated mtDNA methylation, leading to improvements in these parameters and restoration of cell viability while reducing apoptosis. In conclusion, mtDNA methylation is involved in the mechanism of contrast-induced RTEC injury, potentially mediated by over-transcription of COX I and III, abnormal mtROS production, and subsequent mitochondrial damage and dysfunction. Inhibiting mtDNA methylation can provide protective effects against contrast - induced RTEC injury by reducing ROS (mtROS) production.