We aimed to explore the different protective effects of tirofiban on myocardial ischemia-reperfusion (IR) injury in New Zealand white rabbits by comparing the results from different administration methods. Fifty New Zealand white rabbits were randomly divided into a sham group (group A, n = 10) and four IR groups (group B, IR group with injection of physiological saline; group C, tirofiban administered through marginal ear vein after reperfusion; group D, tirofiban injected through coronary ostia before reperfusion; group E, tirofiban injected through coronary artery after blood flow restoration; all n = 10). Myocardial IR injury models were prepared in IR groups. An automatic biochemical analyzer (HITACHI 7020, Japan) was applied for testing serum creatine kinase-MB levels. The myeloperoxidase activity, malondialdehyde levels, nitric oxide synthase activity, and nitric oxide (NO) volume were detected 180 minutes after reperfusion. The myocardial apoptosis was identified using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling technique, and the protein expressions of B-cell lymphoma-2, Bcl-2 associated X, and aquaporin-1 were measured through Western blot. The highest and lowest ST-segment resolution among the IR groups was observed in groups E and B, respectively. The creatine kinase-MB levels at 60, 120, and 180 minutes in group E was greatly decreased than in groups B, C, and D. Compared with the sham group, the IR groups demonstrated evidently elevated myeloperoxidase activity, malondialdehyde levels, inducible NOS activity, NO volume, myocardial apoptotic index, and aquaporin-1 expressions; among the IR groups, these indicators were increased and decreased most in groups B and E, respectively. The B-cell lymphoma-2/Bcl-2 associated X ratio in the IR groups were evidently higher than the sham group, with the highest and lowest rate in groups E and B, respectively. Tirofiban injection through coronary artery after blood flow restoration has a better protective effect against myocardial IR injury than tirofiban administration through coronary ostia before reperfusion and tirofiban injection through the auricular vein after reperfusion.