Osteocytes are the most abundant cellular component of bone and have been considered dormant until recent evidence has demonstrated their critical roles in bone homeostasis and endocrine regulation. Until now the location of osteocytes within mineralized bone has restricted experimental access, especially in vivo. Here, intravital bone imaging by two-photon excitation microscopy allowed us to directly visualize the osteocytic lacuno-canalicular system. We demonstrated that sciatic neurectomy causes significant acidification around osteocytic lacunae and enlargement of lacuno-canalicular areas. These results show that two-photon intravital microscopy is useful for analysis of osteocytes in vivo.