Since their first description 20 years ago, single-domain antibody fragments derived from heavy-chain-only antibodies of camelids, the so-called nanobodies, have emerged as reliable recombinant binding molecules for the proteomic research community. The facile identification of antigen-specific nanobodies and their beneficial biochemical properties regarding size, affinity, and stability supported by multiple crystal structures turn those proteins into versatile binders suitable for multiple applications in the field. During the past decade, nanobodies have been shown as highly functional chaperones assisting the crystallization process of challenging proteins and large protein complexes. In this article, we provide the background knowledge about nanobodies and review their recent application as crystallization chaperones for the determination of protein structures.