Intracellular serpins are proposed to inactivate proteases released from lysosome‐related organelles into the host cell interior, preventing cell death. Serpinb9 opposes the immune cytotoxic protease, granzyme B, and in a number of settings protects cells against granzyme B‐mediated cell death. Using a knockout mouse line engineered to express green fluorescent protein under the serpbinb9 promoter, we demonstrate that serpinb9 is vital for host survival during Ectromelia virus infection by maintaining both mature natural killer NK) cells, and activated CD8⁺ T cells. Serpinb9 expression parallels granzyme B expression within both populations during infection. Maturing serpinb9‐null NK cells exhibit higher levels of granzyme B‐mediated apoptosis during infection; hence there are fewer mature NK cells, and these cells also have lower cytotoxic potential. Thus the serpinb9–granzyme B axis is important for homeostasis of both major cytotoxic effector cell populations.