Objective To investigate whether dexmedetomidine(Dex) preconditioning protects myocardium against ischemia/reperfusion injury(I/RI) through inhibiting endoplasmic reticulum stress(ERS) in rats. Methods Forty-five male rats, weighing 230-270g, were randomly divided into three groups(n=15 for each group): group sham operation(group S), group I/R and group Dex+I/R(group D+I/R). MI/RI model was established by occlusion of left anterior descending coronary artery(LAD) for 30min followed by 6h reperfusion. Dexmedetomidine(1μg‧kg-1‧h-1 for 10min,followed by 0.7μg‧kg-1‧h-1 for 15 min) was infused via the right jugular vein. The ischemic and infracted region size was measured by Evans blue and 2,3,5-triphenyl tetrazolium chloride(TTC) double staining. The mRNA level of endoplasmic reticulum stress related molecules GRP78, CHOP and inflammatory factors IL-1β, IL-6 were detected by real-time quantitative PCR(RT-qPCR). Western blotting was adopted to detected to expression of GRP78, CHOP and cleaved caspase 3. Results Compared with group S, the infract size in group I/R was significantly increased(P<0.05), the mRNA level of GRP78, CHOP, IL-1β, IL-6 and the protein level of GRP78, CHOP and cleaved caspase 3 in group I/R was obviously enchanced(P<0.05). Compared with Group I/R, the above related factors in group D+I/R were significantly declined (P<0.05). Conclusions Our study indicates that dexmedetomidine preconditioning effectively inhibited endoplasmic reticulum stress during myocardial ischemia/reperfusion injury in rats, alleviating apoptosis and inflammation.