In order to identify functional connections in living neuronal networks, it is indispensable to develop flexible and precise stimulation method at the single-cell level to trigger action potentials in neurons. Here we demonstrated femtosecond laser-induced stimulation of a single neuron in dissociated culture of hippocampal neurons. Cultured hippocampal neurons loaded with a fluorescent calcium indicator fluo-4/AM were exposed to femtosecond laser pulses with a center wavelength of 800 nm and pulse duration of ~100 fs through a microscope objective. When a femtosecond laser was focused on a single neuronal cell, fluorescence intensity immediately increased at the laser spot, indicating that intracellular Ca²⁺ concentration increases transiently due to the femtosecond laser irradiation. Following the elevation of Ca²⁺ concentration in a target neuron, an increase in fluorescence intensity of other neuronal cell close to the target neuron was observed, inferring that propagation of the neuronal activity triggered by the elevation of intracellular Ca²⁺ concentration is induced by the femtosecond laser irradiation. Our method can be applicable to functional analysis of living neuronal networks without the use of any chemicals.