Demethylzeylasteral (Dem), the key component of Tripterygium wilfordii Hook F, has been considered to be a traditional Chinese medicine for the treatment of rheumatic diseases. In this study, a mouse colitis model induced by dextran sulfate sodium (DSS) was constructed to clarify the role of Dem in the inflammatory bowel model (IBD). Our results showed that Dem attenuates symptoms of IBD in mice, as evidenced by shortened colon length and weight loss, accompanied by a decrease in the proportion of Th17 cells. Moreover, Dem was shown to inhibit the activation of JAK2 and STAT3. In the IBD mouse model, we used siRNAs targeting either JAK2 or STAT3 to explain the anti-inflammatory effect of Dem. Knockdown of either JAK2 or STAT3 abolished the Th17 inhibition of Dem in vitro. Studies have found that Dem has a significant therapeutic effect on CIA by inhibiting the proliferation and activation of Th17 cells, which further verifies the anti-inflammatory mechanism of Dem. These results suggest that Dem exerts anti-inflammatory effects by inhibiting the differentiation and activation of Th17 cells. Its mechanism may be related to the regulation of the JAK2-STAT3 signaling pathway. Our study will provide a theoretical basis for the application of Tripterygium wilfordii Hook F.