The objective of the present study was to investigate whether pretreatment with single low loading dose of tongxinluo (TXL), a traditional Chinese medicine, 1 h before myocardial ischemia could attenuate no-reflow and ischemia-reperfusion injury by regulating endothelial nitric oxide synthase (eNOS) via the PKA pathway. In a 90-min ischemia and 3-h reperfusion model, minipigs were randomly assigned to the following groups: sham, control, TXL (0.05 g/kg, gavaged 1 h before ischemia), TXL + H-89 (a PKA inhibitor, intravenously infused at a dose of 1.0 μg·kg(-1)·min(-1) 30 min before ischemia), and TXL + N(ω)-nitro-L-arginine (L-NNA; an eNOS inhibitor, intravenously administered at a dose of 10 mg/kg 30 min before ischemia). TXL decreased creatine kinase (CK) activity (P < 0.05) and reduced the no-reflow area from 48.6% to 9.5% and infarct size from 78.5% to 59.2% (P < 0.05), whereas these effects of TXL were partially abolished by H-89 and completely reversed by L-NNA. TXL elevated PKA activity and the expression of PKA, Thr(198) phosphorylated PKA, Ser(1179) phosphorylated eNOS, and Ser(635) phosphorylated eNOS in the ischemic myocardium. H-89 repressed the TXL-induced enhancement of PKA activity and phosphorylation of eNOS at Ser(635), and L-NNA counteracted the phosphorylation of eNOS at Ser(1179) and Ser(635) without an apparent influence on PKA activity. In conclusion, pretreatment with a single low loading dose of TXL 1 h before ischemia reduces myocardial no-reflow and ischemia-reperfusion injury by upregulating the phosphorylation of eNOS at Ser(1179) and Ser(635), and this effect is partially mediated by the PKA pathway.