Calpain activation contributes to the development of infection-induced diaphragm weakness, but the mechanisms by which infections activate calpain are poorly understood. We postulated that skeletal muscle calcium-dependent phospholipase A₂ (cPLA₂) is activated by cytokines and has downstream effects that induce calpain activation and muscle weakness. We determined whether cPLA₂ activation mediates cytokine-induced calpain activation in isolated skeletal muscle (C2C12) cells and infection-induced diaphragm weakness in mice. C2C12 cells were treated with the following: 1) vehicle; 2) cytomix (TNF-α 20 ng/ml, IL-1β 50 U/ml, IFN-γ 100 U/ml, LPS 10 μg/ml); 3) cytomix + AACOCF3, a cPLA₂ inhibitor (10 μM); or 4) AACOCF3 alone. At 24 h, we assessed cell cPLA₂ activity, mitochondrial superoxide generation, calpain activity, and calpastatin activity. We also determined if SS31 (10 μg/ml), a mitochondrial superoxide scavenger, reduced cytomix-mediated calpain activation. Finally, we determined if CDIBA (10 μM), a cPLA₂ inhibitor, reduced diaphragm dysfunction due to cecal ligation puncture in mice. Cytomix increased C2C12 cell cPLA₂ activity (P < 0.001) and superoxide generation; AACOCF3 and SS31 blocked increases in superoxide generation (P < 0.001). Cytomix also activated calpain (P < 0.001) and inactivated calpastatin (P < 0.01); both AACOCF3 and SS31 prevented these changes. Cecal ligation puncture reduced diaphragm force in mice, and CDIBA prevented this reduction (P < 0.001). cPLA₂ modulates cytokine-induced calpain activation in cells and infection-induced diaphragm weakness in animals. We speculate that therapies that inhibit cPLA₂ may prevent diaphragm weakness in infected, critically ill patients.