High consumption of fruits and vegetables has been suggested to provide some protection to smokers who are exposed to an increased risk of numerous cancers and other degenerative diseases. Carrot is the most important source of dietary β-carotene. Therefore, the objective of this study was to investigate whether carrot juice supplementation to smokers can protect against lymphocyte DNA damage and to compare the effect of supplementation of capsules containing purified β-carotene or a placebo (simple lactose). The study was conducted in a randomized and placebo-controlled design. After a depletion period of 14 days, 48 smokers were supplemented with either carrot juice (n = 18), purified β-carotene (n = 16) or placebo (n = 14). Each group was supplemented for 8 weeks with approximately 20.49 mg of β-carotene/day and 1.2 mg of vitamin C/day, as carrot juice (300 ml/day) or purified β-carotene (20.49 mg of β-carotene, 1 capsule/day). Lymphocyte DNA damage was determined using the COMET assay under alkaline conditions and damage was quantified by measuring tail moment (TM), tail length (TL), and% DNA in the tail. Lymphocyte DNA damage was significantly decreased in the carrot juice group in all three measurements. The group that received purified β-carotene also showed a significant decrease in lymphocyte DNA damage in all three measurements. However, no significant changes in DNA damage was observed for the placebo group except TM ( P = 0.016). Erythrocyte antioxidant enzyme was not significantly changed after supplementation. Similarly plasma lipid profiles were not different after carrot juice, β-carotene and placebo supplementation. These results suggest that while the placebo group failed to show any protective effect, carrot juice containing beta-carotene or purified β-carotene itself had great antioxidative potential in preventing damage to lymphocyte DNA in smokers.