The aim of this study was to investigate the tolerance of Clostridium butyricum (C. butyricum) to adverse unsuitable environment in the process of pellet and in the digestive tract of broilers by simulating the condition of pellet and the environment of the digestive tract of broilers in vitro. The tolerance of C. butyricum to high temperature, high pressure, artificial gastric juice and intestinal digestive enzyme were evaluated, respectively. High temperature tolerance test:the experimental diet which contained C. butyricum was treated for 2.5, 5.0 and 7.5 min at 85℃, respectively, and the control group had no treatment. High pressure tolerance test:the experimental diet which contained C. butyricum was treated for 5 min at 0, 0.20, 0.30 and 0.40 MPa, respectively, and the control group had no treatment. Artificial gastric juice tolerance test:the experimental diet which contained C. butyricum was treated for 48 min in artificial gastric juice (pH=2.00, 3.00 and 4.00, respectively) and phosphate buffered saline (pH=2.00, as control group), respectively. Intestinal digestive enzyme tolerance test:the experimental diet which contained C. butyricum was firstly treated for 48 min in artificial gastric juice (pH=3.00), and then treated for 198 min in intestinal digestive enzyme juice and phosphate buffered saline (pH=7.00, as control group), respectively. Each group had three replicates. After the test, the viable count of C. butyricum was measured by plate counting method, and the livability of C. butyricum was calculated. The results showed that the livability of C. butyricum was 70.43%, 52.69% and 46.35% by treating for 2.5, 5.0 and 7.5 min at 85℃, respectively. After treatment at 0.20, 0.30 and 0.40 MPa for 5 min, the livability of C.butyricum was 64.38%, 87.14% and 101.74%, respectively. The livability of C. butyricum was 113.27%, 123.07% and 78.52% by treating for 48 min in artificial gastric juice with the pH of 2.00, 3.00 and 4.00, respectively. The livability of C. butyricum was 47.71% after digestive enzyme juice treatment for 198 min. The results indicate that C. butyricum can tolerate high temperature and high pressure environment in feed processing, and has a better tolerance for artificial gastric juice, but has a lower tolerance for intestinal digestive enzyme.