Hexavalent chromium [Cr(VI)] and its compounds have extensive applications in many industries and are widely known to cause occupational diseases as well as carcinogenic effects in humans. Mitochondrial damage, which is important in Cr(VI)‑induced cytotoxicity, may be characterized by the opening status of the permeability transition pore, the maintenance of the mitochondrial membrane potential and the level of malondialdehyde. The formation of DNA‑protein crosslinks (DPCs) in target tissues appears to be the direct and primary genotoxic effect of Cr(VI) exposure, and the lymphocytic DPCs may be viewed as a biomarker of internal Cr(VI) accumulation. It is well known that vitamin C (vit C) is an important biological reducing agent in humans and animals, which is capable of reducing Cr(VI). Regardless of the evidence from cell culture and in vivo experiments of the protective effect of the antioxidant, vit C, following exposure to Cr(VI), no studies have been conducted to date to demonstrate the time‑order effects of vit C on Cr(VI)‑induced mitochondrial damage and DPC formation. In the present study, by using peripheral blood lymphocytes from Sprague‑Dawley rats, we demonstrated that vit C pre‑ and co‑treatment have a protective effect against Cr(VI)‑induced loss of cell viability and mitochondrial damage, while only vit C co‑treatment has a protective effect against the Cr(VI)‑induced increase in DPCs. The mechanistic investigation revealed that cellular reactive oxygen species levels are correlated with Cr(VI)‑induced mitochondrial damage, and that p53 expression is correlated with the Cr(VI)‑induced increase in DPCs. We concluded that vit C exerts different time‑order effects on Cr(VI)‑induced mitochondrial damage and DPC formation, and that biomarkers, including DPC and p53, may be used in the assessment of the development of Cr(VI)‑induced cancer. These findings facilitate more detailed follow‑up of the Cr(VI)‑exposure populations for secondary prevention.