Objective To explore the effect and mechanism of Xiaoshixionghuangsan in regulating NO levels to protect ischemic myocardium in mice. Methods C57BL/ 6J mice were randomly divided into Blank, sham, model (AMI), Xionghuang (XH), Xiaoshi (XS) and Xiaoshixionghuang ( XSXH) groups. Following establishment of the mouse myocardial infarction model, Blank, sham operation, and model groups were given normal saline, whereas the other groups were given corresponding intervention drugs. Serum levels of cardiac troponin I (cTnI), creatine kinase isoenzyme (CK-MB), and lactate dehydrogenase ( LDH) were detected by ELISA at 12, 24 and 36 h after modeling. On the fourteenth day after modeling, total NO contents in serum were detected with an NO kit, pathological damage of the infarcted mouse myocardium was detected by hematoxylin and eosin staining, and the infarcted area was measured by 2,3,5-triphenyltetrazolium chloride staining. At 12 h and 14 days after Xiaoshixionghuang San administration, serum alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, creatinine, and urea nitrogen were detected by staining. Results Serum levels of cTnI, CK-MB and LDH in the XSXH group were significantly lower compared with AMI, XH and XS groups (P<0. 05). Serum NO levels in the sham group were high, but were significantly decreased in the AMI group (P<0. 05). Compared with the AMI group, NO levels in mice with myocardial infarction were significantly increased in XH and XS groups ( P< 0.01). Compared with single XS or XH, NO contents in the XSXH group were significantly increased ( P< 0. 001). Compared with the AMI group, myocardial infarct sizes in the XSXH group were significantly reduced (P<0. 05). In addition, the decoction markedly improved the degree of pathological damage to the myocardium in mice. Compared with the Blank group, indexes of liver and kidney function in the XH group were significantly increased (P<0. 01), and there was no significant difference in these indexes in the XSXH group (P>0. 05). Conclusions When the classical pathway L-Arginine-eNOS-NO pathway is inhibited during myocardial infarction, Xiaoshixionghuang San can regulate the level of NO in mice through the NO₃^--NO₂^--NO pathway to elicit a significant protective effect on ischemic mouse myocardium without obvious toxic effects on liver or kidney function.