Emerging evidence shows that the transient receptor potential vanilloid 4 (TRPV4) channel is involved in fibrosis in many organs. However, its role in diabetic cardiac fibrosis remains unclear. Our aim was to evaluate the expression level of TRPV4 in the diabetic heart and clarify its role in diabetes-induced cardiac fibrosis. A diabetic animal model was induced by a single intraperitoneal injection of streptozotocin into Sprague-Dawley rats. We also investigated cardiac fibroblasts isolated from neonatal Sprague-Dawley rats. TRPV4 expression was significantly upregulated in both diabetic myocardium and cardiac fibroblasts cultured in high-glucose medium. Masson's trichrome staining revealed that the TRPV4 antagonist HC067047 attenuated the diabetes-induced cardiac fibrosis. Furthermore, HC067047 reduced collagen Ι synthesis and suppressed the transforming growth factor beta 1 (TGF-β1) level as well as the phosphorylation of Smad3 in the diabetic heart. In addition, the TRPV4 antagonist inhibited the proliferation of cardiac fibroblasts, collagen Ι synthesis, and activation of the TGF-β1/Smad3 signaling pathway induced by high-glucose culture medium. Our findings demonstrate that the upregulation of TRPV4 expression mediates diabetic cardiac fibrosis via activation of the TGF-β1/Smad3 signaling pathway.