The aim of this study was to investigate the effects of the clinostat-simulated weightlessness on biological characteristics of MLO-Y4 osteocyte-like cells. MLO-Y4 cells were incubated for 24 h, then randomly divided into 3 groups and rotated in a clinostat as a model of simulated weightlessness for 12 h, 24 h and 48 h. The morphology, cytoskeleton, and secretion of soluble molecules of MLO-Y4 cells were observed and detected. The results show that clinostat culture affects the number of dendrites/cell, cytoskeleton distribution, and secretion of nitric oxide and prostaglandin E2 in MLO-Y4 cells. These results may provide some clue to explore the cellular mechanism of bone loss caused by weightlessness.