This study examined the therapeutic potential of Gardenia fructus and Uncaria rhynchophylla mixture (GU) on thioacetamide (TAA)-induced liver fibrosis in mice. The optimal ratio of GU was determined using LX-2 cells for animal experiments. Liver fibrosis was induced over eight weeks by increasing the TAA doses (100, 200, and 400 mg/kg, i.p.), with the concurrent daily oral administration of silymarin and GU extract (GUL, 50 mg/kg; GUH, 100 mg/kg). Fibrosis-related mRNA expression was analyzed using real-time PCR and immunofluorescence staining to evaluate the anti-fibrotic effects. In addition, serum analysis, Western blotting, and histochemical staining were conducted. Using LX-2 cells, the real-time PCR and immunofluorescence staining results determined the optimal ratio of Gardenia fructus to Uncaria rhynchophylla to be 1:2. The considerably elevated levels of GOT, GPT, bile acids, and ammonia caused by the TAA treatment were significantly lowered with GUH administration. Furthermore, GUH suppressed the reactive oxygen species (ROS) and ONOO − levels in both the serum and liver tissues, as well as the malondialdehyde levels in liver tissue. GU inhibited the protein expression of NADPH oxidase, which generated ROS. GU inhibited NF-κB activation, resulting in a significant decrease in the inflammatory proteins such as Cox-2 and IL-1β. Moreover, the fibrogenic-related factors were effectively reduced by the GU treatment. These findings suggest that GU is a therapeutic agent for alleviating the progression of liver fibrosis.