Endogenous hydrogen sulfide (H2S) was involved in myocardial ischemia postconditioning and alleviated ischemia-reperfusion (I/R) injury, and exogenous H2S postconditioning has a protective effect on the isolated heart I/R injury. Myocardial I/R can induce endoplasmic reticulum stress (ERS), and excessive ERS will induce apoptosis and increase the myocardial injury. Recently, studies have shown that ATF6 is activated in myocardiaocytes by ER stress. In the present study, we determined whether H2S post-conditioning protection myocardium from I/R injury involved in ATF6 activated ERS. Sprague-Dawley rats (male, 250-300g) were anaesthetised with urethane (1g kg-1 i.p.) and were randomly divided into 5 groups (n= 12 in each): Sham group, I/R group, I/R+NaHS groups (H2S post-conditioning group), I/R+TUDCA (Tauro Ursodesoxy Cholic Acid, an ERS blocker) group and I/R+TUDCA+NaHS group. NaHS 14μmol/kg and TUDCA 25mg/kg were injected i.p. after 30min of myocardial ischemia and within reperfusion 10 min. The myocardial I/R rats were subjected to ischemia by 30 min of coronary artery branch of left anterior descending occlusion followed by 2 h of reperfusion. At the end of the reperfusion, myocardial infarct size was examined by triphenyltetrazolium chloride (TTC) staining, serum concentration of H2S was determined with a spectrophotometer, myocardial apoptosis was detected by TUNEL and the protein levels of ATF6, GRP78, PDI, and CHOP were assayed by western blotting. At the end of reperfusion, the average myocardial infarct size was 37.41±3.3%. Compared with the Sham group, the H2S concentration in the serum of the I/R group was significantly decrease (24.98±0.69 vs 44.56±0.86μmol/L, P<0.01); the myocardiaocytes apoptotic rate were significantly increased (84.28±3.03% vs 0.50±0.29%, P<0.01); the endoplasmic reticulum stress related proteins ATF6, GRP78, PDI and CHOP expression level in myocardial tissue were significantly increased (P<0.01). Compared with I/R group, serum H2S concentrations of I/R+NaHS group were significantly increased (43.48±2.31 vs 24.98±0.69μmol/L, P<0.01). Administration of exogenous H2S into rat /H2S post-conditioning could decrease myocardial infarct size (18.69±1.87% vs 37.41±3.3%, P<0.01) and myocardiocytes apoptotic rate (34.23±2.92% vs 84.28±3.03%, P<0.01) significantly. The decrease of ATF6, PDI, GRP78 and CHOP protein level in I/R could be partly reversed by H2S post-conditioning (P<0.05). Using TUDCA to block ER stress could also attenuate I/R injury; compared with I/R+TUDCA group, I/R+NaHS group had even stronger protecting effect on myocardial infarct size and apoptosis rate. This finding suggested that cardioprotection of H2S postconditioning may involve inhibiting apoptosis caused by ATF6 activated excessive ERS induced by myocardiac I/R.