AbstractIschaemia in the heart is accompanied by the accumulation of long‐chain acylcarnitines (LCACs) which is one of the multiple factors that contribute to the ischaemia–reperfusion damage development. Long‐term pre‐treatment that decreases carnitine and LCAC contents also reduces ischaemia–reperfusion (IR) damage; however, the duration of the post‐treatment effects is not known. The aim of the study was to assess the post‐treatment effects of the carnitine transport (OCTN2) inhibitor, methyl‐GBB, on LCAC content and the duration of its cardioprotective effect. Male Wistar rats received methyl‐GBB (5 mg/kg for 28 days), and the anti‐infarction effects on Langendorff‐perfused hearts and the acylcarnitine profile in cardiac tissues were measured up to 28 days following the end of the treatment. Methyl‐GBB pre‐treatment for 28 days decreased LCAC heart tissue content by 87%, and the infarct size was decreased by 57%. Fourteen days post‐treatment, the LCAC content was still decreased by 69%, and the infarct size was decreased by 32% compared to Control. A significant Pearson correlation (r = 0.48, p = 0.026) was found between infarct size and LCAC tissue content in the methyl‐GBB‐treated rat hearts. The addition of 2 mM carnitine to isolated heart perfusate significantly diminished the methyl‐GBB‐induced decrease in LCACs and infarct size. In conclusion, the anti‐infarction effect of methyl‐GBB continues for at least 2 weeks post‐treatment. No less than a 70% decrease in LCAC content is required to protect ischaemic heart tissues, and the decrease in LCAC levels defines the duration of the post‐treatment cardioprotective effect of the OCTN2 inhibitor, methyl‐GBB.