In this study, male mice were treated with fermented and unfermented Lactobacillus plantarum, Lactobacillus bulgaricus, and Lactobacillus rhamnosus black wolfberry juice (10 mL/kg/day) for 40 days, and their prophylactic effects on ulcerative colitis (UC) induced by dextran sodium sulfate were investigated. The intervention of black wolfberry juice reduced the levels of pro-inflammatory cytokines and increased the content of anti-inflammatory cytokines in the serum and colon. In addition, the pathological changes in colon tissue were alleviated, the expression of Bcl-2 protein in the colon was enhanced, and the intestinal microbiota of the mice was regulated, with an increase in Bacteroidetes and a decrease in Helicobacter. These results suggested that black wolfberry juice had an anti-UC function and Lactobacillus fermentation enhanced the anti-inflammatory effect of black wolfberry juice by modulating the intestinal microbiota.