Microglia, the immune cells of the central nervous system, are important for tissue homeostasis and in response to disease. Additionally, they are crucial during brain development, promote synapse elimination and survival, thereby even contributing to higher brain functions like cognition. Microglia are highly motile cells that continuously survey the brain parenchyma by extending and retracting their fine processes, yet the molecular mechanisms governing their dynamic shape changes are not fully understood.The cytoskeletal proteins actin depolymerizing factor and cofilin 1 (ADF/Cfl1) have established significance in neuronal development, function, and cell cycle control through their role in actin filament‘s reorganization. However, their role in microglia remains unexplored.We generated a mouse line with an inducible conditional Adf/Cfl1 knockout in microglia. Knockout of ADF/Cfl1 was proven to be effective on genomic, transcriptional and protein level and resulted in reduced density of microglia in vivo. Besides this, knockout of Adf/Cfl1 also resulted in significant changes in cellular morphology in vivo and ex vivo. Monitoring microglia migration towards a laser-lesion was affected in absence of ADF/Cfl1 in microglia. Knockout of ADF/Cfl1 also resulted in accumulation of filamentous actin. Analysis of gene expression revealed distinct differences of gene regulation after knockout of ADF/Cfl1.These findings demonstrate that ADF/Cfl1 are necessary for morphology, motility and function of microglia.