Th17 cells play an important role in the immune imbalance and inflammatory state in colonic mucosa of Inflammatory Bowel Disease (IBD) and to clarify the mechanism that affect the differentiation of Th17 cells will help us find a new target for the treatment of IBD. MiR-155 which is reported to have an important role in regulating immune system function is also detected to be significantly up-regulated in colonic tissues of IBD patients. However, whether and how miR-155 affects the differentiation of Th17 cells in the colon of IBD patients is still worth studying. Here, we investigated the role of miR-155 in TNBS-induced rat colitis. Firstly, we found that the disease activity index (DAI) and Colon pathological changes were significantly reduced (P < 0.05) by using miR-155 inhibition sequences delivered by lentiviral vector, which revealed that miR-155 inhibition ameliorated TNBS-Induced experimental colitis. Then, we carried out flow cytometry, ELISA, qRT-PCR, and found that in TNBS+miR-155 inhibition group, the proportion of Th17 cells in spleens and mesenteric lymph nodes (MLNs) and the level of the Th17 cell-associated cytokines IL-6, IL-17A, IL-17F and IL-21 in colon tissues were significantly reduced (P < 0.05), which revealed that miR-155 inhibition regulated the differentiation and function of Th17 cells. Finally, we discovered that Jarid2 was significantly elevated (P < 0.05) by miR-155 inhibition and notch1 expression was inversely correlated with Jarid2 by using Immunohistochemistry and western blot. This study suggests that miR-155 inhibition ameliorates TNBS-induced colitis by regulating the Th17 cells differentiation and function and Jarid2/notch1 is closely related with the process.