ABSTRACT Borrelia burgdorferi is the causative agent of Lyme disease, an infectious disease that primarily affects the skin, nervous system, and joints. Uptake of borreliae by immune cells is decisive for the course of the infection, and remodelling of the host actin cytoskeleton is crucial in this process. In this study, we showed that the actin‐regulatory formin Daam1 is important in Borrelia phagocytosis by primary human macrophages. Uptake of borreliae proceeds preferentially through capture by filopodia and formation of coiling pseudopods that enwrap the spirochetes. Using immunofluorescence, we localized endogenous and overexpressed Daam1 to filopodia and to F‐actin‐rich uptake structures. Live‐cell imaging further showed that Daam1 is enriched at coiling pseudopods that arise from the macrophage surface. This filopodia‐independent step was corroborated by control experiments of phagocytic cup formation with latex beads. Moreover, siRNA‐mediated knockdown of Daam1 led to a 65% reduction of borreliae‐induced filopodia, and, as shown by the outside–inside staining technique, to a 50% decrease in phagocytic uptake of borreliae, as well as a 37% reduction in coiling pseudopod formation. Collectively, we showed that Daam1 plays a dual role in the phagocytic uptake of borreliae: first, as a regulator of filopodia, which are used for capturing spirochetes, and second, in the formation of the coiling pseudopod that enwraps the bacterial cell. These data identify Daam1 as a novel regulator of B. burgdorferi phagocytosis. At the same time, this is the first demonstration of a role for Daam1 in phagocytic processes in general.—Hoffmann, A.‐K., Naj, X., Linder, S. Daam1 is a regulator of filopodia formation and phagocytic uptake of Borrelia burgdorferi by primary human macrophages. FASEB J . 28, 3075–3089 (2014). www.fasebj.org