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Chromium picolinate is a popular nutritional supplement whose safety has been questioned because of the potential risk of oxidative DNA damage. To investigate this possibility, a dose-dependent study was performed in piglet hepatocyte cultures in which low (8 μM), medium (200 μM), and high (400 μM) doses of chromium picolinate were tested and compared to untreated controls. After 48 h incubation, there were no significant differences in the levels of intracellular reactive oxygen species, medium lactate dehydrogenase activity, and comet indicators between the three experimental groups and controls (p > 0.05). In the 8 μM-treated group, the intracellular malondialdehyde content was significantly decreased relative to controls (p < 0.05). All of the studied parameters showed a dose-dependent increase that was statistically significant between the low and high doses (p < 0.05). These results suggest that: (1) chromium picolinate may affect the oxidative status of piglet hepatocytes; (2) the appropriate dose (approximately physiological concentration) of chromium picolinate can inhibit lipid peroxidation, and (3) high doses of chromium picolinate have no significant effects on oxidative damage in piglet hepatocytes, but the existing evidence also imply that exposure to a higher dose appears to be unwarranted.