Lipid peroxidation and ferroptosis are critically for the development of cardiac ischaemia-reperfusion (I/R) injury. The proteasome complex is crucial for regulating inflammation and cardiac I/R injury. Proteasome-activating peptide 1 (PAP1) is an activator of the proteasome β5i subunit, but its role in cardiac I/R injury remains unknown. Our results indicate that the administration of PAP1 highly enhanced the expression and activity of the β5i in cardiac tissues possibly by inhibiting of STAT3. Moreover, compared with vehicle control, administration of PAP1 in wild-type mice greatly reversed the I/R-induced decline in myocardial contractility and increases in myocardial infarction, fibrosis, myocyte apoptosis, ROS production and inflammatory response. RNA sequencing revealed that PAP1 mainly affected the genes that were associated with heart contraction, ferroptosis, apoptosis, ROS production and p53. Furthermore, PAP1 clearly decreased the p53 protein and increased the protein levels of SLC7A11 and GPX4 both in mice and cultured cardiomyocytes . Conversely, these protective actions of PAP1 were significantly eliminated in the mice treated with the β5i inhibitor epoxomicin or in the cardiomyocytes transfected with shRNA-β5i but were enhanced by the inhibition of p53 with pifithrin-α. Mechanistically, PAP1 increased the β5i expression, which then bound to p53 and promoted its degradation, resulting in the upregulation of SLC7A11 and GPX4 proteins and the attenuation of oxidative stress and ferroptosis. In summary, our findings suggest that PAP1 can protect against myocardial I/R injury possibly via the β5i-p53-SLC7A11 axis and represent a novel drug candidate for the treating ischaemic heart injury.