Ionized calcium binding adaptor molecule 1 (Iba1) is a microglia/macrophage‐specific calcium‐binding protein. Iba1 has the actin‐bundling activity and participates in membrane ruffling and phagocytosis in activated microglia. In order to understand the Iba1‐related intracellular signalling pathway in greater detail, we employed a yeast two‐hybrid screen to isolate an Iba1‐interacting molecule and identified another actin‐bundling protein, L‐fimbrin. In response to stimulation, L‐fimbrin accumulated and co‐localized with Iba1 in membrane ruffles induced by M‐CSF‐stimulation and phagocytic cups formed by IgG‐opsonized beads in microglial cell line MG5. L‐fimbrin was shown to associate with Iba1 in cell lysate of COS‐7 expressing L‐fimbrin and Iba1. By using purified proteins, direct binding of Iba1 to L‐fimbrin was demonstrated by immunoprecipitation, glutathione S‐transferase pull‐down assays and ligand overlay assays. The binding of Iba1 was also found to increase the actin‐bundling activity of L‐fimbrin. These results indicate that Iba1 forms complexes with L‐fimbrin in membrane ruffles and phagocytic cups, and suggest that Iba1 co‐operates with L‐fimbrin in modulating actin reorganization to facilitate cell migration and phagocytosis by microglia.