OBJECTIVE：To optimi ze the ratio of four comp onents of Compound renshen jianti formulation （Panax ginseng ， Dioscorea oppositifolia ，Lycium barbarum fruit，Alpinia oxyphylla ），and to investigate its anti-fatigue activity and acute toxicity. METHODS：The water extract of Compound renshen jianti formulation was prepared by water extraction ，concentration and decompression drying. By single factor tests ，using weight-bearing swimming time as index ，the effects of four factors were investigated，such as the amount of P. ginseng ，D. oppositifolia ，L. barbarum fruit，A. oxyphylla . On the basis of single factor tests，using comprehensive score of weight-bearing swimming time ，serum urea nitrogen content ，liver glycogen content and AUC of blood lactate after exercise as index ，the formulation was optimized by Box-Behnken response surface method. The mice was divided into blank control group （water），positive control group （Renshen hongjingtian capsules ，0.135 g/kg）and compound low-dose，medium-dose and high-dose groups [the optimal ratio of Compound renshen jianti formulation extract （called“optimal compound formulation ”for short ）4.08，8.16，12.24 g/kg，by crude drug] ，intragastric administration of drug or distilled water 20 mL/kg，once a day ，for consecutive 30 d. The weight-bearing swimming time ，the contents of serum urea nitrogen ，liver glycogen and blood lactate AUC after exercise were used to optimize its anti-fatigue activity of optimal compound formulation. The comprehensive score was calculated based on the measured data of mice in the compound formulation middle-dose group ， and the difference between it and the theoretical prediction value was compared. The mice were given optimal compound formulation intragastrically （total dose 16.00 g/kg， by extract）. The general state ， body mass change ， toxic characteristics and death of mice were observed and recorded for 14 days. Median lethal dose （LD50）and maximum tolerated dose （MTD）were measured. RESULTS ：The optimal formulation ratio of Compound renshen jianti formulation included that P. ginseng 1.5 g，D. oppositifolia 10 g，L. barbarum fruit 10 g，A. oxyphylla 3 g. Results of anti-fatigue activity validation test showed that the optimal compound formulation could significantly prolonged weight-bearing swimming time ，reduced serum content of urea nitrogen ，blood lactate content and its AUC （except for low-dose group ），while significantly increased the content of liver glycogen （P＜0.05 or P＜0.01）. Average comprehensive score of medium-dose group was 96.95，which was only 0.06％ different from the theoretical prediction value of 97.01. The results of acute toxicity test showed that there was no death in mice. The oral MTD of the optimal compound formulation was more than 15 g/kg，which was non-toxic. CONCLUSIONS ：The optimal Compound renshen jianti formulation has effective anti-fatigue activity of mice ，and has no significant toxic effect.