Aims This study investigated the role of L‐3‐n‐Butylphthalide (NBP) in cardiac protection. Methods The left anterior descending coronary arteries (LAD) of the rats were occluded for 30 min following by 2‐h reperfusion to make the ischaemia/reperfusion models. Neonatal cardiomyocytes were cultured and subjected to hypoxia. L‐3‐n‐Butylphthalide was administered intraperitoneally 2 h before the surgery and right after the reperfusion in the in vivo experiments or added to the culture medium in vitro. Haemodynamic parameters were recorded to evaluate the cardiac functions, triphenyltetrazolium chloride (TTC) and Evens blue staining were used to determine the area of risk and infarct area, apoptotic cell numbers were counted with terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining. Western blotting was used to determine the apoptotic protein levels and immune staining to determine the translocation of Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) protein. Results Our research showed for the first time that L‐3‐n‐Butylphthalide had great effects in improving cardiac hemodynamic function and decreasing cardiac infarct areas and apoptotic cell numbers in the peri‐infarct areas. The apoptotic signals investigation showed that L‐3‐n‐Butylphthalide affected the mitochondrial pathway including Bcl‐2 protein expression, inhibition of caspase 3 activation and cytochrome C releasing. Besides, Glyceraldehyde‐3‐phosphate dehydrogenase protein translocation was inhibited by L‐3‐n‐Butylphthalide treatment, and this effect was mediated by endogenous reactive oxygen species (ROS). Conclusion L‐3‐n‐Butylphthalide protects cardiomyocytes from ischaemia/reperfusion‐induced apoptosis by antioxidant effect and affecting mitochondrial apoptotic pathway.