The T300A substitution in ATG16L1 associated with Crohn's disease impairs autophagy, yet up to 50% of humans are heterozygous for this allele. Here, we demonstrate that heterozygosity for the analogous substitution in mice (Atg16L1(T316A)), but not homozygosity, protects against lethal Salmonella enterica Typhimurium infection. One copy of Atg16L1(T316A) was sufficient to enhance cytokine production through inflammasome activation, which was necessary for protection. In contrast, two copies of Atg16L1(T316A) inhibited the autophagy-related process of LC3-associated phagocytosis (LAP) and increased susceptibility. Macrophages from human donors heterozygous for ATG16L1(T300A) displayed elevated inflammasome activation while homozygosity impaired LAP, similar to mice. These results clarify how the T300A substitution impacts ATG16L1 function and suggest it can be beneficial to heterozygous carriers, providing an explanation for its prevalence within the human population.