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We demonstrate intracellular manipulation of synaptic vesicles in living neurons by optical trapping. When an infrared trapping laser is focused on synapses of a neuronal cell labeled with a fluorescent endocytic marker, fluorescence is observed at the focal spot. The fluorescence spectrum is attributed to fluorescent dye in the synaptic vesicles, indicating excitation by two-photon absorption of the trapping laser. The fluorescence intensity increases gradually within ∼100 s of laser irradiation, suggesting that trapping force causes vesicles assembly at the focus. Our method can be applied to manipulate synaptic transmission of a particular neuron in a neuronal network.