Objective To explore whether dexmedetomidine (DEX) can alleviate exertional heatstroke (EHS)-induced rhabdomyolysis (RM) in rats by activating adrenergic α2 receptors, and to explore its potential mechanism based on the reactive oxygen species (ROS)/NOD-like receptor protein 3 (NLRP3)/interleukin-1β (IL-1β) pathway. Methods Thirty-six male Sprague-Dawley (SD) rats, after a 7-day acclimatization training, were randomly divided into six groups: control group (CN group), EHS group, low-dose DEX group (EHS+low DEX group), high-dose DEX group (EHS+high DEX group), DEX combined with yohimbine (YOH) group (EHS+high DEX+YOH group), and YOH group (EHS+YOH), with six rats in each group. Before modeling, EHS+high DEX+YOH group and EHS+YOH group were intraperitoneally injected with YOH at 1 mg/kg, while the other four groups were injected intraperitoneally with an equal dose of physiological saline (0.9% NS). During modeling, except for CN group, the other 5 groups of rats were subjected to heat exercise in a high-temperature and high-humidity chamber to construct an EHS rat model. After successful modeling, EHS+low DEX group was intraperitoneally injected with DEX at 10 μg/kg, EHS+high DEX group and EHS+high DEX+YOH group were intraperitoneally injected with DEX at 30 μg/kg, and CN group, EHS group and DEX+YOH group were intraperitoneally injected with equal doses of saline. After 6 h of observation, all rats were anesthetized, and their blood from the abdominal aorta and gastrocnemius muscle tissue were taken. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of serum tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β and myoglobin (MB) in rats; biochemical assay kit was used to measure the level of creatine kinase (CK) in rat serum; HE staining was used to observe pathological changes in rat gastrocnemius muscle tissues; transmission electron microscopy was used to observe ultrastructural changes in gastrocnemius muscle; 2′,7′-dichlorofluorescent yellow diacetate (DCFH-DA) fluorescent probe was used to detect the level of reactive oxygen species (ROS); and Western blotting was performed to detect the expression levels of NOD-like receptors 3 (NLRP3), aspartic protease-1 (caspase-1) and adrenergic α2A receptor (ADRA2A). Results Compared with CN group, the levels of serum IL-6, IL-1β, TNF-α, CK and MB in EHS group rats were significantly elevated (P<0.01). HE staining results revealed that the gastrocnemius muscle tissues of rats in EHS group had these pathological manifestations such as disarray of muscle fibrous structure, hemorrhage, edema, and infiltration of inflammatory cells. Transmission electron microscopy results showed that the ultrastructure of the gastrocnemius muscle in EHS group exhibited myofibroblasts with swelling and enlarging in size, cytoplasmic vacuolization, and mitochondria with obvious swelling, degranulation, and disappearance of double cristae. Compared with CN group, the expression levels of ROS, NLRP3, and caspase-1 in gastrocnemius of rats in EHS group significantly increased (P<0.01); Compared with EHS group, the levels of TNF-α, IL-6, IL-1β, CK, MB and the expression levels of ROS, NLRP3, caspase-1 in gastrocnemius tissue of rats in EHS+low DEX group and EHS+high DEX group decreased in a dose-dependent manner (P<0.05), and the pathological damage observed with HE staining and transmission electron microscopy was alleviated by DEX. After YOH pretreatment, compared with the EHS+high DEX group, the serum levels of TNF-α, IL-6, IL-1β, CK, MB and ROS, NLRP3 and caspase-1 in the gastrocnemius muscle tissue of rats in EHS+high DEX+YOH group relatively increased (P<0.05), and the pathological damage observed with HE staining and transmission electron microscopy was exacerbated. The expression of ADRA2A in gastrocnemius muscle of EHS group significantly decreased compared with CN group (P<0.01), and the expression of ADRA2A in muscle of rats in DES+low DEX group and EHS+high DEX group was higher than that in EHS group (P<0.05). Conclusions DEX can alleviate EHS-induced RM by activating ADRA2A, potentially through inhibiting the ROS-dependent NLRP3/IL-1β inflammatory pathway.