The present study aimed to investigate the association between myocardial cell apoptosis and calpain-1/caspase-3 expression in a rat model of hypoxic-ischemic brain damage (HIBD). A total of 64 newborn rats were divided into control (n=8; sacrificed on day 7) and HIBD groups (n=56). HIBD group rats were sacrificed 2, 12 or 24 h, or 2, 3, 5 or 7 days following HIBD (n=8/group). A terminal deoxynucleotidyl transferase dUTP nick-end labeling assay was performed to detect myocardial apoptotic cells and calculate the apoptosis index (AI), reverse transcription-polymerase chain reaction was performed to detect myocardial calpain-1/caspase-3 mRNA expression levels and a western blot analysis was conducted to detect calpain‑1 protein expression levels. The correlations between calpain‑1 and caspase‑3 expression levels and AI were analyzed. The results demonstrated that apoptotic myocardial cells in the HIBD groups were markedly increased compared with the control group, with AI peaking in the day 3 group. Caspase‑3 and calpain‑1 mRNA expression levels were increased from 2 and 12 h following HIBD, respectively, with the most elevated levels in the day 2 group. Compared with the control group, calpain‑1 protein expression levels were increased from 2 h, with the greatest expression levels in the day 3 group (P<0.05). Calpain‑1 mRNA and protein (76/80 kDa) expression levels demonstrated positive linear correlations with AI (r=0.786, P=0.001; and r=0.853, P=0.001, respectively) Caspase-3 mRNA expression levels were positively correlated with AI (r=0.894; P=0.001). In conclusion, the present study demonstrated that in rats with HIBD, there is a positive correlation between increased apoptosis of myocardial cells and expression levels of calpain-1 and caspase-3.