Aim To observe the effect of Klotho gene modified bone marrow mesenchymal stem cells (BMSC) transplantation on myocardial fibrosis in rats with heart failure. Methods Isolation, culture and amplification of rat bone marrow mesenchymal stem cells. Klotho transfection to BMSC and RT-PCR detection of Klotho mRNA expression in BMSC. The rat model of chronic heart failure were randomly divided into normal group, saline group, EGFP-BMSC group and EGFP-Klotho-BMSC group, with 6 rats in each group. The cardiac function was detected by Doppler ultrasound after 28 days of cell transplantation. The survival and distribution of the cells were observed by fluorescence microscope. The pathological changes of myocardial tissue were observed by HE staining. Masson staining was used to detect the content of myocardial collagen deposition and the degree of myocardial fibrosis. Results Klotho combined with BMSC treatment can better inhibit myocardial fibrosis, reduce myocardial interstitial collagen deposition and improve cardiac function. Conclusion Klotho gene combined with bone marrow mesenchymal stem cells could attenuate cardiomyocytes hypertrophy and restrain collagens hyperplasy in the progression of myocardial fibrosis.