The research of mitochondrial dysfunction is of great importance and implicated in a range of neurodegenerative diseases. Traditionally, to investigate mitochondrial dynamics and functions, mitochondria are usually stimulated indirectly by treating cells with exogenous chemicals like oxidative agents. Such treatment lacks precision and controllability, and will simultaneously activate unknown complex cell processes. In this study, we report that two-photon 100-μs line scan by a femtosecond laser can induce restorable fragmentation or swelling of any targeted mitochondria instead of ablation or disruption. It can be defined by a customized two-photon line scan and inserted into any microscopy sequence as a single frame. The mitochondrial response is dependent on the peak power of laser pulses, cellular oxidative environment, and membrane permeability transition pores of mitochondria. The translocation of cytochrome C and Bax can be regulated by the photostimulation. Moreover, significant upregulation of Bcl-2 can be observed if the whole cell is stimulated. Those results suggest the mitochondrial and molecular response to photostimulation is quite complex. This femtosecond-laser stimulation method can thus provide a very noninvasive, precise, and controllable method to stimulate single target mitochondria for related biological research.