The adverse effects of anti-tuberculosis (TB) drugs in the intestines were related to alteration of the intestinal microbiota. However, there was less information about microbial metabolism on the adverse reactions. This study aimed to explore whether Lactobacillus casei could regulate gut microbiota or short-chain fatty acids (SCFAs) disorders to protect intestinal adverse reactions induced by isoniazid (H) and rifampicin (R). Male Wistar rats were given low and high doses of Lactobacillus casei two hours before daily administration of anti-TB drugs. After 42 days, colon tissue and blood were collected for analysis. The feces at two-week and six-week were collected to analyze the microbial composition and the content of SCFAs in colon contents was determined. Supplementation of Lactobacillus casei increased the proportion of intestinal goblet cells induced by H and R (p < 0.05). In addition, HR also reduced the level of mucin-2 (p < 0.05), and supplementation of Lactobacillus casei restored. After two weeks of HR intervention, a decrease in OTUs, diversity index, the abundance of Bacteroides, Akkermansia, and Blautia, and an increase of the abundance of Lacetospiraceae NK4A136 group and Rumencoccus UCG-005, were observed compared with the control group (p all < 0.05). These indices in Lactobacillus casei intervention groups were similar to the HR group. Six-week intervention resulted in a dramatic reduction of Lacetospiraceae NK4A136 group, butyric acid, valeric acid and hexanoic acid, while an increase of Bacteroides and Blautia (p all < 0.05). Pretreatment with Lactobacillus casei significantly increased the content of hexanoic acid compared with HR group (p < 0.05). Lactobacillus casei might prevent intestinal injury induced by anti-tuberculosis drugs by regulating gut microbiota and SCFAs metabolism.