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The aim of this study was to investigate the role of MKX in ligament\/tenogenic differentiation of bone marrow derived mesenchymal stem cells (BMMSCs). Human BMMSCs were treated with 50\u2009ng\/ml BMP\u201012 or transduced with MKX or scleraxis (SCX) adenoviral vector. Gene expression analysis was performed by quantitative reverse transcribed polymerase chain reaction (qRT\u2010PCR). Rat BMMSCs were seeded in a collagen scaffold and transplanted into a rat Achilles tendon defect model. Tenogenesis related gene expressions and histological features were analyzed. BMP\u201012 induced tenogenesis in BMMSCs as indicated by increased COL1a1, TNXB, DCN and SCX mRNA, and MKX expression increased simultaneously. Rat BMMSCs enhanced defect repair and were still detectable 3 weeks after transplantation. Increased expressions of COL1a1, TNC and TNMD in vivo were also correlated with upregulated MKX. Adenoviral MKX promoted expression of COL1a1, TNXB, and TNMD in BMMSCs. This study demonstrated that MKX gene expression is enhanced during the tenogenic differentiation of BMMSCs in vitro and in vivo, and the adenoviral overexpression of MKX increases tendon extracellular matrix gene expression and protein production. Thus, MKX is a key factor for tenogenic differentiation of MSCs. 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. 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