Tumor necrosis factor (TNF)-alpha is a key cytokine regulator of bone and mediates inflammatory bone loss. The molecular signaling that regulates bone loss downstream of TNF-alpha is poorly defined. Recent studies implicated an important role of microRNAs (miRNAs) in TNF-alpha-mediated bone metabolism, including osteoblasts differentiation, osteoclasts differentiation and apoptosis. However, there are very few studies on the complex regulation of miRNAs during TNF-alpha-induced osteoblasts apoptosis. In the present study, the clonal murine osteoblastic cell line, MC3T3-E1, was used. We screened for differentially expressed miRNAs during TNF-alpha induced MC3T3-E1 cell apoptosis and identified microRNA-23a as a potential inhibitor of apoptosis. To delineate the role of microRNA-23a in apoptosis, we respectively silenced and overexpressed microRNA-23a in MC3T3-E1 cells. We found that microRNA-23a depletion significantly enhances TNF-alpha-induced MC3T3-E1 cell apoptosis and over-expressing microRNA-23a remarkably attenuates this phenomenon. Mechanistic studies showed that microRNA-23a inhibits Fas expression through a microRNA-23a-binding site within the 3′-untranslational region of Fas. The post-transcriptional repression of Fas was further confirmed by luciferase reporter assay. These results showed that microRNA-23a, an important protecting factor, plays a significant role in the process of TNF-alpha induced MC3T3-E1 cell apoptosis, by regulating Fas expression. J. Cell. Biochem. 114: 2738–2745, 2013. © 2013 Wiley Periodicals, Inc.