Cytolytic CD8(+) T cells (CTLs) kill virally infected cells, tumor cells, or other potentially autoreactive T cells in a calcium-dependent manner. To date, the molecular mechanism that leads to calcium intake during CTL differentiation and function has remained unresolved. We demonstrate that desmoyokin (AHNAK1) is expressed in mature CTLs, but not in naive CD8(+) T cells, and is critical for calcium entry required for their proper function during immune response. We show that mature AHNAK1-deficient CTLs exhibit reduced Ca(v)1.1 alpha1 subunit expression (also referred to as L-type calcium channels or alpha1S pore-forming subunits), which recently were suggested to play a role in calcium entry into CD4(+) T cells. AHNAK1-deficient CTLs show marked reduction in granzyme-B production, cytolytic activity, and IFN-gamma secretion after T cell receptor stimulation. Our results demonstrate an AHNAK1-dependent mechanism controlling calcium entry during CTL effector function.