The imbalance of follicular helper T (Tfh) cell subset is closely related to the occurrence of inflammatory bowel disease (IBD). Ginsenoside Rg1 (G-Rg1), the main component of ginseng, has excellent immunomodulatory and anti-inflammatory effects. Here, mice colitis was induced by dextran sulfate sodium and treated with G-Rg1 (200 mg/kg/day) for 14 days. Results showed that G-Rg1 can significantly alleviate symptoms of colitis in mice, regulate the balance of Tfh cell subsets, promote the secretion of IL-4 and IL-10, and inhibit the expression of IFN-γ, IL-17A, and IL-21. Molecular docking analysis revealed that G-Rg1 has excellent binding activity with target genes of the TLR/MyD88 signaling pathway, and and can reduce the expression levels of proteins such as TLR2, MyD88, IRAK4, TRAF6, and TAK1. These findings suggest that G-Rg1 can effectively regulate the balance of Tfh cell subsets, and its potential mechanism is related to the inhibition of the TLR/MyD88 signaling pathway.