Ulcerative colitis is one of the most common sorts of inflammatory bowel disease. This study investigates the protective effects of thymoquinone against sodium dodecyl sulfate (SDS)-induced intestinal damage and elucidates the underlying mechanisms using the Drosophila melanogaster model of ulcerative colitis. We found that Drosophila fed thymoquinone from larval to adult stages were resistant to SDS injury in adulthood. Thymoquinone pretreatment significantly restored the abnormal behaviors and intestinal morphological defects in Drosophila exposed to SDS. Moreover, thymoquinone protected the intestinal barrier function by inhibiting the overactivated c-Jun N-terminal kinase (JNK) pathway in the intestine induced by SDS. Further studies indicated that thymoquinone inhibits the JNK pathway by reducing intestinal reactive oxygen species (ROS) levels. This research provides novel pathological and mechanistic insights into the potential application of thymoquinone in developing functional foods or natural medicines, highlighting its significance in treating ulcerative colitis.