Abstract: Pyruvate kinase is one of the important glycolytic enzymes that affects the rate of postmortem muscle glycolysis and then regulates meat quality. The study investigated the effects of protein phosphorylation and the crosstalk between protein phosphorylation and acetylation on pyruvate kinase activity, clarified the regulating mechanism of pyruvate kinase phosphorylation and acetylation on its enzymatic activity, in order to provide a theoretical basis for the development of fresh meat quality regulation technology. Protein kinase A (PKA) and alkaline phosphatase (AP) were added to construct the phosphorylation model of different phosphorylation levels. The groups were set as PKA treatment group, AP treatment group, and control group (C). The samples were incubated at 4 ℃ for 0, 2, 12 and 24 h, an equal amount of acetyltransferase was added to each treatment group after 24 h incubation to regulate the acetylation level of pyruvate kinase to construct the model of phosphorylation and acetylation interaction, which were incubated in vitro for 0, 2, 12 and 24 h under the same conditions. The phosphorylation level, acetylation level, pyruvate kinase activity, pyruvate content, phosphorylation site, and secondary structure changes of pyruvate kinase were analyzed. The results showed that in the phosphorylation model, the phosphorylation level and enzyme activity of pyruvate kinase in PKA group were significantly higher than that in the AP and C groups at 0 to 12 h incubation (P<0.05), indicating that phosphorylation promoted pyruvate kinase activity. In the crosstalk model of phosphorylation and acetylation, the phosphorylation level of pyruvate kinase in PKA and C groups were significantly reduced at 24 h incubation (P<0.05), indicating that acetylation inhibited the phosphorylation level of pyruvate kinase. There was no significant change of pyruvate kinase acetylation level in PKA group, and the acetylation level was increased gradually in AP group, indicating that the phosphorylation of pyruvate kinase inhibited its acetylation level. The acetylation level of pyruvate kinase in AP group was significantly higher than that of PKA group, and its enzyme activity was significantly lower than that of PKA group (P<0.05), indicating that acetylation could inhibit pyruvate kinase activity. After phosphorylation of pyruvate kinase, the secondary structure was changed from disorder to order, and the phosphorylation of pyruvate kinase at Ser249 reduced the total structural energy, increased the bond energy, and enhanced the structural stability of pyruvate kinase. The results of this study indicated that phosphorylation of pyruvate kinase promoted its enzymatic activity, helping to improve the structural stability of pyruvate kinase, and there might be an antagonistic crosstalk of pyruvate kinase phosphorylation and acetylation.