Carotenoids are important components of plant antioxidant systems, which protect photosystems from photooxidative destruction during ultraviolet-B (UV-B) exposure. The influence of carotenoids on total antioxidant capacity (TAC) of plants has rarely been studied. In this study, tobacco (Nicotiana tabacum L., 'K326') seedlings exposed to UV-B radiation were used in order to evaluate the effects of ambient levels of UV-B radiation on carotenoid accumulation. The aim was to investigate whether carotenoids could enhance TAC as a means of UV protection. Our results showed that leaf carotenoid content in the low UV-B exposure (+9.75 μW/cm2) plants was approximately 8% higher than that observed in control plants at 2-8 days of exposure. At high UV-B exposure (+20.76 μW/cm2), the carotenoid content increased rapidly after 1 day's exposure (10.41% higher than the control), followed by a return to the content as in control plants. Furthermore, carotenoid content positively correlated with TAC (P = 0.024). These results suggest that carotenoids have antioxidant properties and play an important role in the antioxidant system. UV-B exposure increased the carotenoid synthesis capability of plants. The plants could deplete the carotenoids to scavenge excess ROS at high UV-B radiation levels, which protects the tobacco plant from oxidative damage caused by UV-B stress.