Objective To study the effect and mechanism of recombinant human brain natriuretic peptide (rh-BNP) in alleviating myocardial ischemia-reperfusion (I/R) injury by regulating mitogen activated protein kinase (MAPK) pathway. Methods A total of 128 adult male Sprague-Dawley (SD) rats with specific pathogen free were selected. The SD rats were divided into groups according to random number table, including, sham operation (Sham) group, I/R group, I/R+rh-BNP group, negative control adenovirus (Ad-NC)+Sham group, Ad-NC+I/R group, Ad-NC+I/R+rh-BNP group, p38 mitogen-activated protein kinase adenovirus (Ad-p38MAPK)+I/R group and Ad-p38MAPK+I/R+rh-BNP group, with 16 SD rats in each group. Myocardial I/R injury model was established by ligation of left anterior descending coronary artery. Before modeling, rh-BNP was injected intraperitoneally or adenovirus was injected into myocardium; 180 minutes after reperfusion, the contents of lactate dehydrogenase (LDH), creatine kinase isoenzyme (CK-MB) in serum, myocardial infarction size, the contents of reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α) and the expression of phosphorylated p38MAPK (p-p38MAPK), phosphorylated JNK (p-JNK) and phosphorylated extracellular regulated protein kinases 1/2 (p-ERK1/2) were detected. Results The contents of LDH, CK-MB, myocardial infarction size, the contents of TNF-α, ROS and the expression of p-p38MAPK and p-JNK in I/R group were higher than those in Sham group, p-ERK1/2 expression level was lower than that in Sham group (P<0.05). The contents of LDH, CK-MB, myocardial infarction size, the contents of TNF-α, ROS and the expression of p-p38MAPK in I/R+rh-BNP group were lower than those in I/R group (P<0.05), the expression of p-JNK and p-ERK1/2 had no significant difference compared with I/R group (P>0.05). The contents of LDH, CK-MB, myocardial infarction size, the contents of TNF-α, ROS and the expression of p-p38MAPK in Ad-p38mapk+I/R+rh-BNP group were higher than those in Ad-NC+I/R-rh-BNP group (P<0.05). Conclusion rh-BNP can alleviate myocardial I/R injury, which is related to inhibiting p38MAPK pathway, reducing inflammation response and oxidative stress response.