In order to control callus browning and provide theoretical and technical basis for tissue culture of Cyclocarya paliurus, the effects of three antioxidants on the callus growth, browning and physiological and biochemical characteristics were studied. The results showed that an appropriate concentration of antioxidants could effectively restrain callus browning rate, promote callus growth and prevent the increase of cytoplasmic membrane permeability in the process of cultivation. Among the three antioxidants, vitamin C (VC) was the most effective, and phytic acid (PA) came next. The optimal concentration was 100 mg/L for VC, 250 mg/L for PA, and 50 -100 mg/L for citric acid (CA).The growth rate of callus fresh weight with 100 mg/L VC treated group increased by 73.88%, and browning rate and membrane permeability decreased by 56.52% and 45.75%, respectively as compared with the control and the difference was statistically significant. The appropriate concentration of antioxidants could inhibit PAL activity in callus to some degree, while it had no obvious effect on PPO activity. Altogether, it was suggested that appropriate concentrations of three antioxidants could effectively reduce the callus browning, promote the growth. The regulatory effects may be associated with PAL activity. The regulation of PPO activity may not be the main pathway of browning control by antioxidants. The mechanism of PPO regulation need to be further studied.