Microgravity (microXg) leads to a 10-15% loss of bone mass in astronauts during space flight. Osteoclast (OCL) is the multinucleated bone-resorbing cell. In this study, we used the NASA developed ground-based rotating wall vessel bioreactor (RWV), rotary cell culture system (RCCS) to simulate microXg conditions and demonstrated a significant increase (2-fold) in osteoclastogenesis compared to normal gravity control (Xg). Gene expression profiling of RAW 264.7 OCL progenitor cells in modelled microXg by Agilent microarray analysis revealed significantly increased expression of critical molecules such as cytokines/growth factors, proteases and signalling proteins, which play an important role in enhanced OCL differentiation/function. Transcription factors such as c-Jun, MITF and CREB implicated in OCL differentiation are upregulated; however no significant change in the levels of NFATc1 expression in preosteoclast cells subjected to modelled microXg. We also identified high-level expression of calcium-binding protein, S100A8 (calcium-binding protein molecule A8/calgranulin A) in preosteoclast cells under microXg. Furthermore, modelled microXg stimulated RAW 264.7 cells showed elevated cytosolic calcium (Ca(2+)) levels/oscillations compared to Xg cells. siRNA knock-down of S100A8 expression in RAW 264.7 cells resulted in a significant decrease in modelled microXg stimulated OCL differentiation. We also identified elevated levels of phospho-CREB in preosteoclast cells subjected to modelled microXg compared to Xg. Thus, modelled microXg regulated gene expression profiling in preosteoclast cells provide new insights into molecular mechanisms and therapeutic targets of enhanced OCL differentiation/activation to prevent bone loss and fracture risk in astronauts during space flight missions.