Ever studies have limited to investigate the dynamic behavior and molecular mechanisms on cultured cell lines, and little is known about the behavior of mitochondria in vivo. Here we found that mitochondria are dynamic organelles in skeletal muscle CX-4945 fibers in vivo for the first time with mtPAGFP, in spite of their compact and “crystal-like” arrangement between bundles of myofilaments. These mitochondria dynamically communicated with each other through nanotunneling-mediated fusion, allowing them to exchange mitochondrial matrix contents manifested by the propagation of mtPAGFP. However, the propagation of mtPAGFP was blunted in HFD-induced mice, indicating that the dynamic behavior was inhibited in obese mice. This change was associated with the decrease of mitochondrial fusion proteins and increase of mitochondrial fission proteins. Finally, decreased mitochondrial dynamics was accompanied with impaired mitochondrial oxygen consumption and ATP production in HFD-induced mice. Although mitochondrial dynamics has been described and extensively studied in C2C12 cells, a cell model used for skeletal muscle study, these results cannot be directly applied to adult skeletal muscle, because both mitochondrial morphology and distribution are quite different between adult skeletal muscle cells and C2C12 cells. A main factor obstructing the advance of the study in mitochondrial dynamics in skeletal muscle is the lack of successful ways for detection of the dynamic behaviors, because it is hard to visualize the dynamics of mitochondria in skeletal muscle with conventional GFP imaging approach as a result of lack of mitochondrial mobility. Using photoactivatible technology, we observed intermitochondrial content transfer in living animals in vivo and directly demonstrated that mitochondria are dynamic organelles in skeletal muscle in real-time. With this approach, we also quantified the rate of mitochondrial communication as well as the detection of mitochondrial content exchange between fused mitochondria. In addition, in vivo imaging allowed us to visualize the real status and activities of mitochondria in living animals, which provides a unique and timely approach for the investigation of mitochondrial dynamics in both physiological and pathophysiological conditions. Although we still did not detect mitochondrial movement in skeletal muscle, a prerequisite for conventional mitochondrial fusion, these mitochondria could communicated with each other by extending filamentous mitochondrial nanotubules—nanotunneling. With this manner, mitochondria can bypass the restriction of myofilament and “talk” with each other even if they are distant. These mitochondrial nanotubules are highly dynamic structures, demonstrated by the fact that the extending mitochondrial filament could fuse with neighboring mitochondria or retract to the main body. This kind of dynamic communication among mitochondria in skeletal muscle may protect the metabolically active cells from injury by preventing the accumulation of detrimental metabolites.