Although functions of autophagy-related (Atg) proteins in canonical autophagy are well understood, recent studies reveal the roles of various Atg proteins in other biological processes. While many of the Atg genes are highly conserved throughout evolution, Atg8 gene has undergone expansion in the genome of higher eukaryotes. Mammalian Atg8 homologues consist of LC3 and Gabarap subfamilies, all of which are involved in canonical autophagy. In contrast, the role of each Atg8 homologue members in non-canonical processes is not fully understood. Here we show a unique role of Gate-16 in an interferon-γ (IFN-γ)-mediated antimicrobial response. Cells lacking Gate-16, but not those lacking Gabarap and Gabarapl1, or LC3a and LC3b, are defective in IFN-γ-induced clearance of vacuolar pathogens such as Toxoplasma. Gate-16, but not LC3b, specifically associates with Arf1 to mediate uniform distribution of IFN-inducible GTPases. Gate-16-deficiency reduces Arf1 activation, leading to formation of IFN-inducible GTPase-containing aggregates, hampering their function. Furthermore, mice lacking Gate-16 alone, either systemically or specifically in the myeloid compartment, are susceptible to Toxoplasma. Thus, Gate-16 is uniquely required for antimicrobial host defense through cytosolic distribution of IFN-inducible GTPases.