Precise actin regulation is essential for diverse cellular processes such as axonal growth, cell migration and endocytosis. twinfilin (twf) encodes a protein that sequesters actin monomers, but its in vivo functions are unclear. In this study, we characterized twf-null mutants in a metazoan for the first time and found that Drosophila twf negatively regulates F-actin formation in subcellular regions of rapid actin turnover in three different systems, namely postsynaptic neuromuscular junction (NMJ) synapses, migratory border cells and epithelial follicle cells. Loss of twf function results 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 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 using the fluorescent dye FM1-43 uptake assay. Furthermore, electrophysiological analysis under high-frequency stimulation showed 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.