Objective Micro-gravity affects the process of bone metabolism and miRNA level of expression. However, the molecular mechanism underlying the alterations of cellular activity and miRNA under the micro-gravity is not fully understood. The aim of this study is to investigate the effect of micro-gravity on MC3T3-E1 cells. Methods The differential miRNA and mRNA expression profiling of MC3T3-E1 cells during exposure to micro-gravity were established by RNA transcriptome sequencing technology (RNA-seq). The RNA sequencing results were validated using quantitative real-time polymerase chain reaction (Q-PCR). Bioinformatic analyses were applied for further study of these differentially expressed miRNAs and mRNAs. Results A total of 1912 coding transcripts and 160 miRNAs were detected along with osteogenic differentiation under conditions of micro-gravity. Bioinformatic analysis revealed 10 core regulatory genes including 7 mRNAs and 3 miRNAs. Based on the analysis and verification, we identified one miRNA, miR-9_6666-5p, which is likely to play important roles in osteogenic differentiation process under micro-gravity. Conclusion The present study showed that the process of osteoblast differentiation was repressed. Our results could lead to a better understanding of the molecular mechanisms of genes and miRNAs, and their cooperation underlying the effects of micro-gravity on osteogenic differentiation and bone formation.