Fascin-1 mediated actin-bundling activity is central to the generation of plasma membrane protrusions required for cell migration. Dysregulation of cellular protrusions is observed in metastatic cancers, where they are required for increased invasiveness, and this is often correlated with overexpression of Fascin-1. Therefore, there is interest in generating therapeutic Fascin-1 inhibitors. We present the identification of Nb 3E11, a nanobody inhibitor of Fascin-1 actin-bundling activity, filopodia formation and cell migration. The crystal structure of the Fascin-1/Nb 3E11 complex reveals the structural mechanism of inhibition. Nb 3E11 occludes an actin-binding site on the third -trefoil domain of Fascin-1 that is currently untargeted by chemical inhibitors and induces a conformational change in the adjacent domains to stabilise Fascin-1 in an inhibitory state similar to that adopted in the presence of small molecule inhibitors. Nb 3E11 can be utilised as a tool inhibitor molecule to aid in the development of Fascin-1 targeted therapeutics.