Heavy ion radiotherapy has unfolded a great promise for cancer therapy. Understanding the cellular response mechanism to heavy ion radiation is required for exploring the measures of overcoming the devastating side effects. Here, we performed a quantitative proteomics to investigate the mechanism of carbon heavy ion irradiation on human AHH-1 lymphoblastoid cells. Totally we identified 4602 proteins and quantified 4569 proteins among which with high coverage on mitochondria. After stringent filtering, we found 290 proteins were significantly up-regulated and 16 proteins were down-regulated. Functional analysis revealed that these up-regulated proteins specifically enriched in the process of DNA damage repair, mitochondrial ribosome and especially mitochondrial respiratory chain, which occupied approximately fifty percent of the accumulated proteins. Bioinformatics analysis and functional study demonstrated that these up- regulated mitochondrial respiratory chain proteins enhanced ATP production and simultaneously ROS releasing. More importantly, the increased ROS led to organelle secondary injury and the lagged DNA double strand breaks. Consistently, the expression of antioxidant enzymes was up-regulated for free radical scavenging. The mechanism of lagged secondary injury originated from disturbing of mitochondrial respiratory chain, the “target” outside nuclear, explored here may provide a novel target for cell self-repairing against heavy ion radiation induced cellular damage.