Significance Guanylate-binding proteins (GBPs) belong to interferon-inducible GTPases and mediate a broad spectrum of innate immune responses against various pathogens. Their protective functions require oligomerization induced by nucleotide binding and/or catalysis, but the actual molecular mechanisms are still elusive. Here, we report the crystal structures of human GBP5 (hGBP5) in both its nucleotide-free state and nucleotide-bound state, as well as nucleotide-free human GBP2 (hGBP2). We show that hGBP5 forms a closed face-to-face dimer upon GTP loading. This closed conformation is crucial to its anti–HIV-1 activity. Furthermore, with hGBP2 structure and SAXS validation, we propose a plausible working model for GTP-induced assembly of GBPs. Our findings lay the foundation to better understand the molecular mechanisms of GBPs and their immune functions.