Objective To observe the effects of dexmedetomidine (Dex) on microtubule‑associated protein 1 light 3 (LC3) and phosphatidy‑linositol 3‑kinase/protein kinase B (PI3K/Akt) signal pathway in isolated rat heart during ischemia‑reperfusion injury, and to explore the protective effects of Dex on myocardium. Methods A total of 48 healthy adult male SD rats (8−10 weeks old and weighing 250−300 g) were used to establish an isolated heart model of ischemia‑reperfusion with Langendorff perfusion device. After successful modeling, a 48 rat hearts were divided into 4 groups (n=12), according to the random number table method: a blank control group (group NC, which was continuously perfused with K‑H solution over 120 min), an ischemia‑reperfusion group (group I/R, which was perfused with K‑H solution over 30 min followed by global ischemia for 30 min, and reperfusion over 60 min), a Dex preconditioning group (group Dex+I/R, which was perfused with K‑H solution over 10 min followed by infusion of K‑H solution containing 25 μg/L Dex for 15 min, elution of K‑H solution for 5 min, ischemia for 30 min, and reperfusion for 60 min), a Dex preconditioning+Warman group (group Dex+I/R+W, which was intraperitoneally injected with 15 μg/kg of Warman 30 min before thoracotomy, along with the similar treatments with group Dex+I/R). Their heart rate (HR), left ventricular diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), the maximum rate of increase or decrease of left ventricular pressure (±dp/dtmax) were recorded at the end of equilibrium (after infusion over 30 min) and reperfusion. The coronary perfusion fluid was collected at the end of reperfusion and the level of lactate dehydrogenase (LDH) was examined by a microplate reader. The myocardiac tissues were taken and myocardial infarct size was measured by 2, 3, 5‑triphenyltetrazolium chloride (TTC) staining, and the percentage of myocardial infarct size was calculated. The expression of autophagosomal marker LC3, Akt, phosphorylated Akt (p‑Akt), mammalian target of rapamycin (mTOR), and phosphorylated mTOR (p‑mTOR) was determined by Western blot. Results Compared with group NC, remarkable decreases in HR, ±dp/dtmax, and LVDP and the levels of p‑Akt and p‑mTOR as well as increases in LVEDP, myocardial infarction size, and the activity of LDH in coronary perfusion fluid, and the level of myocardial LC3 were found in other groups (P<0.05). Compared with group I/R, remarkable increases in HR, ±dp/dtmax, and LVDP and the levels of p‑Akt and p‑mTOR as well as decreases in LVEDP, myocardial infarction size, and the activity of LDH in coronary perfusion fluid, and the level of myocardial LC3 were found in group Dex+I/R (P<0.05). Compared with group Dex+I/R, remarkable decreases in HR, ±dp/dtmax and LVDP and the levels of p‑Akt and p‑mTOR as well as increases in LVEDP, myocardial infarction size, and the activity of LDH in coronary perfusion fluid, and the level of myocardial LC3 were found in group Dex+I/R+W (P<0.05). Conclusions Dex preconditioning can protect ischemia‑reperfusion injury in isolated rat heart, which may be related to decreased autophagy in cardiomyocytes during ischemia reperfusion period through activating PI3K/Akt signal pathway and enhancing downstream mTOR.