Baicalein is a natural flavonoid extracted from the root of Scutellaria baicalensis that exhibits a variety of pharmacological activities. In this study, we investigated the molecular mechanisms underlying the protective effect of baicalein against cardiac hypertrophy in vivo and in vitro. Cardiac hypertrophy was induced in mice by injection of isoproterenol (ISO, 30 mg·kg ·d ) for 15 days. The mice received caudal vein injection of baicalein (25 mg/kg) on 3rd, 6th, 9th, 12th, and 15th days. We showed that baicalein administration significantly attenuated ISO-induced cardiac hypertrophy and restored cardiac function. The protective effect of baicalein against cardiac hypertrophy was also observed in neonatal rat cardiomyocytes treated with ISO (10 μM). In cardiomyocytes, ISO treatment markedly increased reactive oxygen species (ROS) and inhibited autophagy, which were greatly alleviated by pretreatment with baicalein (30 μM). We found that baicalein pretreatment increased the expression of catalase and the mitophagy receptor FUN14 domain containing 1 (FUNDC1) to clear ROS and promote autophagy, thus attenuated ISO-induced cardiac hypertrophy. Furthermore, we revealed that baicalein bound to the transcription factor FOXO3a directly, promoting its transcription activity, and transactivated catalase and FUNDC1. In summary, our data provide new evidence for baicalein and FOXO3a in the regulation of ISO-induced cardiac hypertrophy. Baicalein has great potential for the treatment of cardiac hypertrophy.