The processes involved in the progression of myocardial cells towards hypertrophy and its gradual transition to heart failure represent a multifactorial health disorder. The aim of this study was to identify the molecular mechanism(s) underlying the abnormal overexpression of miR-23b-5p and its involvement in the promotion of cardiac hypertrophy and dysfunction via HMGB2. A type 9 recombinant adeno-associated virus (rAAV9) was employed to manipulate miR-23b-5p expression under conditions of thoracic aortic constriction (TAC)-/angiotensin-II (Ang-II)-induced cardiac dysfunction. Cardiac structures and functions were assessed by echocardiography and invasive pressure-volume analysis. HMGB2 expression under conditions of cardiac hypertrophy was detected by western blotting. The biochemical relationship between miR-23b-5p and HMGB2 was verified using a luciferase reporter vector, lentiviral construct comprising the miR-23b-5p mimic sequence, and microRNA inhibitor (miR-inhibitor). The expression levels of miR-23b-5p were increased in the hearts under conditions of both Ang-II- and TAC-induced cardiac hypertrophy. The results of the luciferase activity analysis showed that HMGB2 is a supposed target of miR-23b-5p. miR-23b-5p overexpression in vivo aggravated pressure overload-induced cardiac hypertrophy and dysfunction, whereas the miR-inhibitor increased HMGB2 expression and reversed these effects. In the present study, we observed that miR-23b-5p mediates and is involved in the aggravation of cardiac hypertrophy and dysfunction via the HMGB2 signaling pathway.