Both mechanical loading and intracellular autophagy play important roles in bone homeostasis; however, their relationship remains largely unexplored. The objectives of this study were to determine whether osteocytes undergo autophagy upon fluid shear stress (FSS) loading and to determine the correlation between mechanically induced autophagy and ATP metabolism. Autophagic vacuoles were observed by transmission electron microscopy (TEM) in osteocyte-like MLO-Y4 cells subjected to FSS. Increased autophagic flux was further confirmed by the increased amount of the LC3-II isoform and the degradation of p62. Fluorescent puncta distributed in the cytoplasm were observed in the GFP-LC3 transformed cells subjected to FSS. Furthermore, FSS-induced ATP release and synthesis in osteocytes were attenuated by inhibiting autophagy with 3-MA. After FSS exposure, a high ratio of cell death was observed in cultures pretreated with 3-MA, an autophagy inhibitor, with no significantly different Caspase 3/7 activity. Our results indicated that FSS induces protective autophagy in osteocytes and that mechanically induced autophagy is associated with ATP metabolism and osteocyte survival. From the clinical perspective, it may be possible to enhance skeletal cell survival with drugs that modulate the autophagic state, and the autophagy-related pathway could be a potential target for the prevention of ageing-related bone disorders.