Increasing evidence shows that miRNAs play pivotal roles in cardiovascular diseases, including heart failure (HF). The aim of this study was to investigate the role of miR-129-5p in chronic heart failure and the underlying mechanisms. The levels of miR-129-5p and HMGB1 in chronic heart failure patients (CHF) and normal controls were examined by RT-qPCR and ELISA. Cardiac function, hemodynamics parameters, oxidative stress, and inflammation factors were analyzed in CHF rat model after transfection of miR-129-5p or HMGB1. Dual-luciferase activity reporter assay was conducted to validate the interaction between miR-129-5p and HMGB1. Downregulation of miR-129-5p and upregulation of HMGB1 were observed in the serum of CHF patients, respectively. Transfection of miR-129-5p improved heart function and hemodynamic parameters, as well as attenuated oxidative stress and inflammation factors in CHF rats. We further confirmed that HMGB1 is a direct target of miR-129-5p. Transfection of miR-129-5p also decreased the mRNA and protein levels of HMGB1 in myocardial tissues of CHF rats. Overexpression of HMGB1 diminished the effects of miR-129-5p on ameliorating oxidative stress and inflammatory response in rats with CHF. Our findings suggest that miR-129-5p protects the heart by targeting HMGB1.