The goal of this study was to elucidate whether there is an increase in myocardial tetrahydrobiopterin (BH4), which is a cofactor for nitric oxide synthase, during the late phase of ischaemic preconditioning (IPC) leading to cardioprotection against myocardial infarction and, if so, to examine the induction mechanisms of BH4 synthesis. Rats were preconditioned with four cycles of 3 min left main coronary artery (LCA) occlusion followed by 10 min reperfusion. Twenty-four hours later, the rats were subjected to 20 min ischaemia by LCA ligation and 2 h reperfusion, and the infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining. The IPC protocol reduced the infarct size, and increased the BH4 content and expression of GTP-cyclohydrolase I (GTPCH), which is the rate-limiting enzyme for BH4 synthesis. Administration of a GTPCH inhibitor attenuated both the reduction in infarct size and the increase in BH4 levels. Moreover, the increase in BH4 content was reduced by administration of catalase or a Janus tyrosine kinase-2 (JAK2) inhibitor. These observations suggest that upregulation of BH4 synthesis in the heart contributes to an acquisition of ischaemic tolerance in late IPC, and the increase in myocardial BH4 content seems to be mediated by the induction of GTPCH via the H2O2–JAK2 pathway.