Mitochondria are dynamic organelles with constantly changing morphologies. Despite recent reports indicating that mechanical cues modulate mitochondrial morphologies and functions, there is a lack of methods that can exclusively and precisely exert mechanical forces to and deform mitochondria in live cells. Therefore, how mitochondria sense and respond to mechanical forces remains largely elusive. Optogenetic methods open up new venues for remote and precise manipulation of intracellular activities using light, providing an unprecedented opportunity to establish targeted mechano-stimulation toward mitochondria. This chapter describes the development of a novel optogenetic approach to optically mechanostimulate and induce the deformation of mitochondria. In this approach, light-gated protein–protein heterodimerization recruits force-generating molecular motors to the outer mitochondrial membrane, enabling direct exertion of mechanical force on mitochondria. Details for the design, application, and experimental procedures are laid out in this chapter. This method presents a mitochondria-specific mechano-stimulator for studying the correlation between mitochondrial morphology and functions as well as mitochondrial mechanobiology.