Membrane fusion is a fundamental cellular process regulating intracellular transport, neurotransmission, enzyme secretion, hormone release, and the entry/exit of viruses, to name a few. Knowledge of how opposing bilayers fuse, besides advancing our understanding of these cellular processes, will provide us with the facts to ameliorate secretory defects and prevent cellular entry or exit of pathogenic viruses. In the last few years, great strides have been made in our understanding of the molecular machinery and mechanism of membrane fusion. In this Special Issue of Cell Biology International, entitled ‘Membrane fusion: machinery and mechanism’, we have tried to cover several aspects of this vital cellular process, providing insights on the machinery, mechanism and dynamics of the process. Membrane fusion studies reported in this Special Issue have been performed on whole cells, synaptic terminals, viruses, and fusion proteins.