Objective To investigate the protective effect and its possible mechanism of yulangsan chalcone (MHBFC) on H9c2 cardiomyocyte hypertrophy induced by angiotensin II (Ang II) based regulating autophagy though AMPK/mTOR signaling pathway. Methods H9c2 cardiomyocytes were divided into blank control group, model group, low-, medium- and high-dose MHBFC groups. After intervention with MHBFC, Ang II (1μmol/L) was used to induce cardiomyocyte hypertrophy model. The number and average surface area of H9c2 cardiomyocytes per unit area were detected. The apoptosis of H9C2 cardiomyocytes was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) method. Mitochondrial membrane potential was detected by mitochondrial membrane potential detection kit (JC-1 fluorescent probe method). The contents of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) were detected by enzyme-linked immunosorbent assay (ELISA). The expression of autophagy marker proteins Beclin1 and p62 was detected by reverse transcription-polymerase chain reaction (RT-PCR). Western blot was used to detect the expression levels of p-AMPK/AMPK, p-mTOR/mTOR and p-ULK1/ULK1 in AMPK/mTOR signaling pathways. Results MHBFC improved the reduction of cardiomyocytes number and cardiomyocyte hypertrophy induced by Ang II (P<0.05 or P<0.01). TUNEL assay showed that MHBFC reduced the apoptosis induced by Ang II (P<0.05 or P<0.01). The results of JC-1 fluorescent probe assay showed that MHBFC alleviated the decrease rate of mitochondrial membrane potential and reduced cell apoptosis induced by Ang II (P<0.05 or P<0.01). The results of ELISA showed that MHBFC reduced the levels of ANP, BNP and β-MHC in cardiomyocytes induced by Ang II (P<0.05 or P<0.01). The results of RT-PCR showed that MHBFC increased the expression of Beclin1 (P<0.05 or P<0.01) and reduced the expression of p62 (P<0.05 or P<0.01). Western blot assay showed that MHBFC increased the protein expressions of p-AMPK/AMPK and p-ULK1/ULK1 (P<0.05 or P<0.01) and down-regulated the protein expression of p-mTOR/mTOR (P<0.05 or P<0.01). Conclusion MHBFC can effectively alleviate H9c2 cardiomyocyte hypertrophy and injury induced by Ang II, and its mechanism may be related to activating AMPK/mTOR signaling pathway, promoting mitophagy, reducing the expression of cardiomyocyte hypertrophy markers and inhibiting mitochondria-mediated apoptosis.