Ionized calcium binding adaptor molecule 1, Iba1, is an EF hand calcium binding protein whose expression is restricted to macrophages/microglia. In this study, Iba1 was shown to colocalize with F-actin in membrane ruffles induced by macrophage colony-stimulating factor and in phagocytic cups formed during zymosan phagocytosis. Expression of mutant Iba1 carrying either N-or C-terminal deletions or carrying a substitution in the calcium binding domain, suppressed the membrane ruffling and the phagocytosis. These results indicate that Iba1 is a key molecule in membrane ruffling and the phagocytosis of macrophages/microglia. Furthermore, Iba1 colocalized with a small GTPase Rac in the membrane ruffles and the phagocytic cups. The Iba1 mutants also suppressed membrane ruffling induced by dominant active Rac1V12, but do not affect microspikes by Cdc42V12 and stress fibers by RhoAV14. These observations suggest that Iba1 is involved in Rac and calcium signaling pathways.