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.