Immunological tolerance is critical for maintaining gut homeostasis. An imbalance between interleukin-17 (IL-17)-producing T helper 17 (T17) cells and regulatory T cells (T cells) is involved in ulcerative colitis (UC) pathogenesis. Dendritic cells (DCs) are able to induce T cell differentiation. Paeoniflorin (PF) is a monoterpene glucoside that is commonly used for treatment of autoimmune disease. However, the immunological mechanism of PF involvement in UC treatment is unclear. The present study aimed to explore whether PF can restore the T17/T balance by modulating DCs. The effects of PF on DCs, T17 cells and T cells were measured. Furthermore, PF-treated DCs were injected into mice with 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. PF inhibited MHC-II and CD86 expression on the DC surface (P < 0.05), decreased interleukin (IL)-12 secretion in vitro and in vivo (P < 0.05), and restored the T17/T ratio in the mouse model of colitis (P < 0.05). PF-treated DCs diminished T17 differentiation (4.26% in vitro and 1.64% in vivo) and decreased IL-17 expression (P < 0.05) while inducing CD4CD25Foxp3 T differentiation (7.82% in vitro and 6.85% in vivo) and increasing Foxp3 and IL-10 production (P < 0.05). Additionally, both PF and PF-treated DCs improved colonic histopathology in the mouse model of colitis (P < 0.05). In conclusion this study suggested that PF can ameliorate TNBS-induced colitis by modulating the DC-mediated T17/T balance.