Neutrophil elastase (NE), elevated in the cystic fibrosis (CF) airway, causes macrophage phagocytic failure. We previously reported that NE increases the release of protease calcium ion-dependent papain-like cysteine protease-2 (Calpain-2) in macrophages. We hypothesized that NE mediates macrophage failure through activation of Calpains. We demonstrate that Calpain inhibition rescued NE-induced macrophage phagocytic failure in murine alveolar macrophages in both cftr-null and wild-type genotypes. We then sought to determine how NE regulates Calpain-2. Human monocyte-derived macrophages (hMDMs) from persons with CF (PwCF) and non-CF subjects were treated with NE or control vehicle, and cell lysates were prepared to evaluate Calpain-2 protein abundance by Western and Calpain activity by a specific activity kit. Calpain is activated by intracellular calcium and inactivated by an endogenous inhibitor, Calpastatin. hMDMs were thus treated with NE or control vehicle and cell lysates were analyzed for increased intracellular calcium by Fluo-4 assay and for Calpastatin protein abundance by Western. NE increased Calpain-2 protein and activity, degraded Calpastatin, and increased intracellular calcium in macrophages. At baseline, there are no differences in Calpain activity, Calpain-2 and Calpastatin expression, and intracellular calcium between CF and non-CF macrophages. NE increased macrophage Calpain-2 protein and Calpain activity by two potential mechanisms: degradation of Calpastatin and/or increased intracellular calcium. In summary, Calpain inhibition restored NE-induced macrophage phagocytic failure suggesting a potential CFTR-independent target for phagocytic failure in the CF airway.NEW & NOTEWORTHY Neutrophil elastase, a cystic fibrosis airway inflammation biomarker, increases macrophage Calpain activity, and Calpain inhibition partially restores the decreased phagocytosis in neutrophil elastase-challenged macrophages. Neutrophil elastase increases Calpain-2 protein, degrades the Calpain inhibitor, Calpastatin, and increases intracellular calcium as potential mechanisms of Calpain activation. This presents a novel mechanism for macrophage dysfunction relevant to cystic fibrosis.