Precise actin regulation is essential for diverse cellular processes such as axonal growth, cell migration, and endocytosis. twinfilin (twf) encodes an actin monomer sequestering protein, but its in vivofunctions are unclear. In this study, we characterized twf null mutants in a metazoan for the first time and found that Drosophila twf negatively regulatesF-actin formation in subcellular regions of rapid actin turnover in three different systems, namely the postsynaptic neuromuscular junction (NMJ) synapses, migratory border cells, and epithelial follicle cells. L oss of twf function resulted in defects in axonal growth in the brain and border cell migration in the ovary. Additionally, we found that the actin dependent postsynaptic localization of the glutamate receptor GluRIIA, but not GluRIIB, was specifically reduced in twf mutants. More importantly, we showed that twf mutations caused significantly reduced presynaptic endocytosis at NMJ synapses as detected by the fluorescent dye FM1-43 uptake assay . Furthermore, electrophysiological analysis under high frequency stimulation showed a compromised neurotransmission in twf mutant synapses, confirming an insufficient replenishment of synaptic vesicles. Together, our results reveal that twinfilin promotes actin turnover in multiple cellular processes that are highly dependent on actin dynamics.