In most cells, mitochondria are highly dynamic organelles that constantly fuse, divide and move. These processes allow mitochondria to redistribute in a cell and exchange contents among the mitochondrial population, and subsequently repair damaged mitochondria. However, most studies on mitochondrial dynamics have been performed on cultured cell lines and neurons, and little is known about whether mitochondria are dynamic organelles in vivo, especially in the highly specialized and differentiated adult skeletal muscle cells. Using mitochondrial matrix-targeted photoactivatable green fluorescent protein (mtPAGFP) and electroporation methods combined with confocal microscopy, we found that mitochondria are dynamic in skeletal muscle in vivo, which enables mitochondria exchange contents within the whole mitochondrial population through nanotunneling-mediated mitochondrial fusion. Mitochondrial network promotes rapid transfer of mtPAGFP within the cell. More importantly, the dynamic behavior was impaired in high-fat diet (HFD)-induced obese mice, accompanying with disturbed mitochondrial respiratory function and decreased ATP content in skeletal muscle. We further found that proteins controlling mitochondrial fusion MFN1 and MFN2 but not Opa1 were decreased and proteins governing mitochondrial fission Fis1 and Drp1 were increased in skeletal muscle of HFD-induced mice when compared to normal diet-fed mice. Altogether, we conclude that mitochondria are dynamic organelles in vivo in skeletal muscle, and it is essential in maintaining mitochondrial respiration and bioenergetics.