Intestinal mucositis is a commonly encountered toxic side effect in patients undergoing 5-fluorouracil (5-FU)-based chemotherapy. Numerous studies have shown that probiotics enable improving chemotherapy-induced intestinal mucositis, but the beneficial effects of probiotics differ depending on the strain. Therefore, in the present studies we suggest that S. thermophilus ST4 separated from raw milk may assess mucoprotective activity in 5-FU-induced intestinal mucositis. In our causal-comparative study design, fifteen mice were randomized assigned into three groups (n = 5/each group): control group, 5-FU group and 5-FU+S. thermophilus ST4 group. The control group was orally administrated saline only, and the 5-FU group was followed by intraperitoneal injection of 5-FU for 3 days after 10-day saline administration, and the 5-FU+S. thermophilus ST4 group was intragastrically subjected for S. thermophilus ST4 once per day during the whole experiment, starting from the first day of the experiment, followed by 5-FU intraperitoneal injection for 3 days after 10-day S. thermophilus ST4 pretreatment. Diarrhea score, pro-inflammatory cytokines serum levels, intestinal histopathology and short chain fatty acid were assessed. Here, we demonstrated the beneficial effects of S. thermophilus ST4 derived from raw milk against 5-FU-induced intestinal mucositis, including body weight reduction, appetite loss and diarrhea. Intrinsically, S. thermophilus ST4 effectively maintained epithelium structure in small intestines and colons as well as reduced the intestinal inflammation. Besides, S. thermophilus ST4 significantly increased the expression of acetic acid, reinforcing the muco-protective effects. In conclusion, our results demonstrate that S. thermophilus ST4 supplementation ameliorates 5-FU-induced intestinal mucositis. This suggests probiotic may serve as an alternative therapeutic strategy for the prevention or management of 5-FU-induced mucositis in the future.