Abstract: In order to study the isolation and identification of Clostridium butyricum in alfalfa silage silo and study its effect on toxicity and growth performance of mice. In this study, Clostridium butyricum was isolated and cultured from silage alfalfa. The suspected strains were identified by morphological, physiological and biochemical and 16 S rDNA sequence analysis. The optimal growth temperature and pH value of positive Clostridium butyricum were analyzed, and then the toxicity and growth performance of mice were tested. Acute oral toxicity test: 20 SPF male Kunming（KM） mice with body weight of 18 to 22 g were randomly divided into control group and Clostridium butyricum group, with 10 mice in each group. The Clostridium butyricum group was gavaged with 2×10¹⁰ cfu/kg Clostridium butyricum isolate diluent and the control group was given equal volume of normal saline by gavage at 9:00 and 15:00 every day. The pre-test lasted for 5 days and the experiment lasted for 10 days. Weighing before the test, denoted as initial weight. After the end of the experiment, the mice were killed by cervical dislocation, weighed and recorded as the final weight. The heart, liver, spleen and kidney were weighed and the coefficients of each organ were calculated. Growth performance test: 120 SPF male KM mice with body weight of 18 to 22 g were randomly divided into control group, low-dose group, medium-dose group, high-dose group and commercial bacteria group with 6 replicates in each group and 4 mice in each replicate. The control group was intragastric administered with normal saline, the low-dose group, medium-dose group and high-dose group were intragastric administered with 2×10~6,2×10~8,2×10¹⁰ cfu/kg Clostridium butyricum isolated diluent, respectively, and the commercial bacteria group was intragastric administered with 2×10~8 cfu/kg Clostridium butyricum at 9:00 and 15:00 every day. The pre-test lasted for 5 days, and the experiment lasted for 28 days. Weighing before the experiment and on the 14 th and 28 th day of the experiment, daily food consumption, diarrhea number and death number were recorded. The average daily gain, average daily feed intake, feed/gain, diarrhea rate and mortality were calculated on 0 to 14 days and 15 to 28 days. The results showed that a strain of Clostridium butyricum, named as strain C3, was successfully isolated and purified from silage alfalfa. The optimum growth temperature of Clostridium butyricum was between 35 ℃ and 40 ℃ and the pH value was about 7.0-8.0. No poisoning or death occurred between the Clostridium butyricum group and the control group, and there were no significant differences in the initial and final body weight, the weight of the heart, liver, spleen, kidney and the coefficient of each organ between the two groups（P＞0.05）. Zero to fourteen days after intragastric administration, there were no significant differences in average daily gain, average daily feed intake and feed/gain among KM mice（P＞0.05）, and the diarrhea rate and death rate were 0; 14-28 days of intragastric administration, compared to the control group, average daily gain in low, medium and high dose groups were significanty increased（P＜0.05）, average daily feed intake were reduced（P＜0.05 or P＞0.05）,and feed/gain were significantly reduced（P＜0.05）. The results indicated that Clostridium butyricum C3 strain isolated from silage alfalfa had the potential to be developed as a green and non-toxic feed additive.