Cardiac fibrosis is one of the pathological characteristics of diabetic cardiomyopathy (DbCM). Matrine treatment has proven to be effective in cases of organ fibrosis and cardiovascular diseases. In the present study, the anti-fibrosis-associated cardioprotective effects of matrine on DbCM were investigated. Rats with experimental DbCM were administered matrine orally. Cardiac functions were evaluated using invasive hemodynamic examinations. Cardiac compliance was assessed in isolated hearts. Using Sirius Red and fluorescence staining, the collagen in diabetic hearts was visualized. MTT assay was used to select non‑cytotoxic concentrations of matrine, which were subsequently used to treat isolated cardiac fibroblasts incubated under various conditions. Western blotting was performed to assess activation of the transforming growth factor‑β1 (TGF‑β1)/Smad signaling pathway. Rats with DbCM exhibited impaired heart compliance and left ventricular (LV) functions. Excessive collagen deposition in cardiac tissue was also observed. Furthermore, TGF‑β1/R‑Smad (Smad2/3) signaling was revealed to be markedly activated; however, the expression of inhibitory Smad (I‑Smad, also termed Smad7) was reduced in DbCM. Matrine administration led to a marked recovery in LV function and heart compliance by exerting inhibitory effects on TGF‑β1/R‑Smad signaling pathway‑induced fibrosis without affecting I‑Smad. Incubation with a high concentration of glucose triggered the TGF‑β1/R‑Smad (Smad2/3) signaling pathway and suppressed I‑Smad signaling transduction in cultured cardiac fibroblasts, which led to an increase in the synthesis of collagen. After cardiac fibroblasts had been treated with matrine at non‑cytotoxic concentrations without affecting I‑Smad, matrine blocked TGF‑β1/R‑Smad signaling transduction to repress collagen production and deposition. In conclusion, the results of the present study demonstrated that TGF‑β1/Smad signaling‑associated cardiac fibrosis is involved in the impairment of heart compliance and LV dysfunction in DbCM. By exerting therapeutic effects against cardiac fibrosis via its influence on TGF‑β1/Smad signaling, matrine exhibited cardioprotective effects in DbCM.