Objective To study the changes in mitochondrial adenosine triphosphate (ATP) sensitive potassium channel Kir6.1 and mitophagy in cardiomyocyte hypertrophy. Methods The primary cardiomyocytes derived from 24-hour-old neonatal rats born were randomly divided into control group and model group. The control group was cultured in a high glucose medium containing 10% fetal bovine serum, while for the model group, 140 μmol/L isoproterenol (ISO) was added to the medium for 48 h to establish a cellular model of cardiomyocyte hypertrophy. The concentration of N-terminal pro brain natriuretic peptide (NT-proBNP) in the culture supernatant of cardiomyocytes was detected by enzyme linked immunosorbent assay. The mRNA expression of atrial natriuretic peptide (ANP) mRNA, brain natriuretic peptide (BNP) mRNA, β-myosin heavy chain (β-MHC) mRNA, inward rectifyimg potassium channel 6.1 (Kir6.1) mRNA and autophagy related gene 8 (Atg8) mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of Kir6.1, microtubule-associated protein 1 light chain 3 (LC3) and FUN14 domain-containing protein 1 (FUNDC1) were detected by Western blotting. GraphPad Prism statistics v9.0.0 was used for statistical analysis. Data comparison between two groups was performed using student′s t test. Results The supernatant concentration of NT-proBNP was significantly higher in the model group than the control group [(1699.43407.01) vs (808.6891.46) pg/ml, P<0.05]. When compared with the control group, the model group had obviously increased ANP, BNP and β-MHC expression and decreased Kir6.1 and Atg8 expression at mRNA level, and reduced expression of Kir6.1 as well as LC3 and FUNDC1 at protein level (all P<0.05). Conclusion The expression of mitochondrial ATP sensitive potassium channel and the level of mitophagy are decreased in cardiomyocyte hypertrophy.