Postmortem glycolysis is directly linked to the incidences of PSE (pale, soft and exudative) and DFD (dark, firm and dry) meats which cause significant loss to meat industry. AMP-activated protein kinase (AMPK) is a major regulator of postmortem glycolysis. However, there are two isoforms of the AMPKα catalytic subunit, and their roles in glycolysis of postmortem muscle remain unclear. The objective was to identify the isoform specific roles of AMPK in postmortem glycolysis. Wild type, AMPKα1, and AMPKα2 knockout (KO) mice were used in the current study. AMPK in Longissimus muscle was activated shortly after death. AMPKα2 but not AMPKα1 KO abolished the activity of AMPK in postmortem muscle. In addition, AMPKα2 KO reduced postmortem pH decline and the generation of lactate, while AMPKα1 KO had no significant effect. Finally, the glycogen content of skeletal muscle was reduced in AMPKα2 KO but not AMPKα1 KO mice. Data clearly demonstrate that AMPKα2 catalytic subunit mainly regulates postmortem glycolysis in muscle.