Objective To detect the levels of IL-1, IL-6,TNF-α and IFN-γ in serum and colon tissue of rat models of ulcerative colitis with spleen and kidney Yang deficiency, and to explore their roles in the pathogenesis of ulcerative colitis (UC). Methods The rat model of ulcerative colitis with Yang deficiency of spleen and kidney was induced by perfusion of rhubarb decoction plus intramuscular injection of hydrocortisone and combined with TNBS (2,4,6-trinitrobenzenesulfonic acid) and ethanol enema. Sixty SPF wistar rats (body weight 180±10 g, male:female=1:1) were randomly divided into blank control group, UC model with spleen kidney Yang deficiency for 7 days, 14 days and 21 days groups, respectively. The levels of IL-1, IL-6, TNF-α and IFN-γ in serum and colon tissue were detected by ELISA. Results Compared with the blank group, the levels of IL-1, IL-6, TNF-α and IFN-γ in serum and colon tissue of rat UC model group with spleen kidney Yang deficiency were greatly increased (P<0.05), especially evidently increased in the model group at 21 days. Conclusions The pro-inflammatory cytokines IL-1, IL-6, TNF-α and IFN-γ play an important role in the pathogenesis of ulcerative colitis with syndrome of spleen and kidney Yang deficiency.