BACKGROUND: Honokiol (HKL), a natural extract of the bark of the magnolia tree and an activator of the mitochondrial protein sirtuin-3 (SIRT3), has been proposed to possess anti-inflammatory effects. This study investigated the inhibitory effects of HKL on T helper (Th) 17 cell differentiation in colitis. METHODS: Serum and biopsies from 20 participants with ulcerative colitis (UC) and 18 healthy volunteers were collected for the test of serum cytokines, flow cytometry analysis (FACS), and relative messenger RNA (mRNA) levels of T cell subsets, as well as the expression of SIRT3 and phosphorylated signal transducer and activator of transcription/retinoic acid-related orphan nuclear receptor γt (p-STAT3/RORγt) signal pathway in colon tissues. In vitro, naïve clusters of differentiation (CD) 4 + T cells isolated from the mouse spleen differentiated to subsets including Th1, Th2, Th17, and regulatory T (Treg) cells. Peripheral blood monocytes (PBMCs) from healthy volunteers were induced to the polarization of Th17 cells. After HKL treatment, changes in T cell subsets, related cytokines, and transcription factors were measured. The dextran sulfate sodium (DSS)-induced colitis and interleukin (IL)-10-deficient mice were intraperitoneally injected with HKL. These experiments were conducted to study the effect of HKL on the development, cytokines, and expression of signaling pathway proteins in colitis. RESULTS: Patients with UC had higher serum IL-17 and a higher proportion of Th17 differentiation in blood compared with healthy participants; while IL-10 level and the proportion of Treg cells were lower. Higher relative mRNA levels of RORγt and a lower SIRT3 expression in colon tissues were observed. In vitro, HKL had little effect on the differentiation of naïve CD4+ T cells to Th1, Th2, or Treg cells, but it downregulated IL-17 levels and the Th17 cell ratio in CD4+ T cells from the mouse spleen and human PBMCs under Th17 polarization. Even with a STAT3 activator, HKL still significantly inhibited IL-17 levels. In DSS-induced colitis mice and IL-10 deficient mice treated with HKL, the length of the colon, weight loss, disease activity index, and histopathological scores were improved, IL-17 and IL-21 levels, and the proportion of Th17 cells were decreased. Sirtuin-3 expression was increased, whereas STAT3 phosphorylation and RORγt expression were inhibited in the colon tissue of mice after HKL treatment. CONCLUSIONS: Our study demonstrated that HKL could partially protect against colitis by regulating Th17 differentiation through activating SIRT3, leading to inhibition of the STAT3/RORγt signaling pathway. These results provide new insights into the protective effects of HKL against colitis and may facilitate the research of new drugs for inflammatory bowel disease.