Objective To explore the effects of a Chinese medicine, Jiuxieling granules, on the expression of TLR4 and NF-κB p65 mRNA and proteins in ulcerative colitis model rats with spleen and kidney yang deficiency. Methods The rat model of ulcerative colitis with spleen and kidney yang deficiency was established by oral administration of rhubarb decoction, intramuscular injection of hydrocortisone and TNBS (2,4,6-trinitrobenzene sulfonic acid) combined with ethanol enema. All the 90 rats were randomly divided into blank control group, UC model of spleen kidney yang deficiency group, and Jiuxieling granules treatment groups for 7 days, 14 days and 21 days, respectively, and positive control group (SASP). After all the rats were killed and colon samples were collected, the expression of TLR4 and NF-κB p65 mRNA and proteins of colon tissues were detected by RT-qPCR and SP immunohistochemical. Results The results of immunohistochemiscal assay indicated that expressions of TLR4 and NF-κB p65 in the UC model group were significantly enhanced, when compared with that of the blank group (P<0.01). After treatment, the expressions of TLR4 and NF-κB p65 in all treatment groups were significantly decreased (P<0.01 or P<0.05). The RT-qPCR assay results indicated that the expressions of TLR4 and NF-κB p65 in the UC model group were significantly enhanced when compared with that of the blank group (P<0.01). After treatment, the expressions of TLR4 and NF-κB p65 in all treatment groups were reduced (P<0.01 or P<0.05). Conclusions Jiuxieling granules can alleviate the mucosal inflammation and facilitate the mucosal repair. The main reasons of which may be associated with reduction of TLR4 and NF-κB p65 gene transcription and protein expression in the ulcerative colitis model rats with spleen and kidney yang deficiency.