To uncover the underlying molecular mechanism of long non-coding RNA in the osteogenic differentiation process of bone marrow mesenchymal stem cells (BMSCs), HOXA transcript at the distal tip (HOTTIP) was selected by using a lncRNA microarray assay. Results showed that HOTTIP was significantly upregulated during osteogenic differentiation of human BMSCs. Downregulation of HOTTIP by shRNA inhibited the osteogenic differentiation of BMSCs. Overexpression of HOTTIP by lentiviral vector promoted human BMSCs osteogenic differentiation by increasing the transcription of beta-catenin. RIP assay and RNA pulldown assay confirmed the interaction between HOTTIP and WDR5, a transcription factor binding to the promoter of beta-catenin. The interaction promoted the translocation of WDR5 into the nucleus and increased the transcription of beta-catenin. Implanted HOTTIP-overexpressing BMSCs increased ectopic bone formation in nude mice. HOTTIP is a conservative long noncoding RNA that is essential for osteogenic differentiation of BMSC. HOTTIP enhances osteogenic differentiation via interaction with WDR5 and up-regulation of beta-catenin gene expression, therefore activating Wnt/beta-catenin signalling pathway. (C) 2020 Elsevier Inc. All rights reserved.