Abstract: Objective To investigate the effect of circRNA_005647 on fibrotic phenotype of mouse cardiac fibroblasts (CFs) and explore its mechanism. Methods We used an angiotensin II (Ang-II) capsule pump (daily dose of 1.46 mg/kg for 2 weeks) to establish a mouse model of myocardial fibrosis in C57BL/6 mice. Masson staining was used to detect myocardial fibrosis in the myocardium. The expression of circRNA_005647 in the myocardium of Ang-II-infused mice and in Ang-II-treated CFs were detected with real-time PCR. Actinomycin D and RNase R exonuclease digestion were used to test the stability of circRNA_005647 in mouse CFs. Over-expression of circRNA_005647 was achieved in the CFs by infecting the cells with a recombinant circRNA_005647 adenovirus (rAd-circRNA_005647), and the expressions of Col1a1, Col3a1 and Acta2 were detected in the cells with real-time PCR and Western blotting. Dual luciferase reporter assay, RNA antisense purification and RNA Pull down assay were performed to identify the interaction between circRNA_005647 and miR-27b-3p. Results CircRNA_005647 was up-regulated in the myocardium of Ang-II- infused mice and in Ang-II-treated mouse CFs (P<0.01). CircRNA_005647 was more stable than its host gene Myosin IXA (Myo9a) in response to actinomycin D (P<0.01) and RNase R exonuclease treatment. The expressions of fibrosis-associated genes was down-regulated in the CFs over-expressing circRNA_005647 (P<0.05). Dual luciferase reporter assay, RNA antisense purification and RNA Pull down assay revealed the interaction between miR-27b-3p and circRNA_005647. MiR-27b-3p obviously enhanced the fibrotic phenotype but reversed the inhibitory effect of circRNA_005647 on the expression of fibrosis associated genes in the CFs (P<0.05). Conclusion CircRNA_005647 is upregulated in cardiac fibrosis and inhibits the expression of fibrosis-related genes through sponging miR-27b-3p in mouse CFs.