Exposure to microgravity causes bone loss in humans, and the underlying mechanism is believed to be at least partially due to a decrease in bone formation by osteoblasts. Here, we examined the hypothesis that microgravity changes osteoblast gene expression profiles, resulting in bone loss. For this study, we developed an in vitro system that simulates microgravity using the Random Positioning Machine (RPM) to study the effects of microgravity on 2T3 pre-osteoblast cells grown in gas-permeable culture disks. Exposure of 2T3 cells to simulated microgravity using the RPM for up to 9 days significantly inhibited alkaline phosphatase activity, recapitulating a bone loss response as seen in real microgravity conditions, without altering cell proliferation and shape. Next, we carried out a DNA microarray analysis to determine the gene expression profile of 2T3 cells exposed to 3 days of simulated microgravity. Among 10,000 genes examined with the microarray, 88 were downregulated while 52 were upregulated significantly by simulated microgravity by more than two-fold in comparison to the static 1g condition. We then verified the microarray data for some of the genes relevant in bone biology by using real-time PCR assays and immunoblotting. We confirmed that microgravity downregulated levels of alkaline phosphatase, runt-related transcription factor 2, osteomodulin, and parathyroid hormone receptor 1mRNAs, upregulated cathepsin K mRNA, and did not significantly affect bone morphogenic protein 4 and cystatin C protein levels. The identification of gravisensitive genes provides a useful insight in generating further hypotheses regarding their roles not only in microgravity-induced bone loss but also in the general population of patients with similar pathologic conditions such as osteoporosis.