To achieve neuronal differentiation of mouse bone mesenchymal stem cells (bMSCs) into neuron-like cells and explore the role of miR-122-5p that may regulate T-box brain 1 (Tbr1) expression during the induction. BMSCs were cultured and induced with butylated hydroxyanisole, retinoic acid (RA), basic fibroblast growth factor, and nerve growth factor in vitro. The cells were stained for neuron-specific enolase (NSE) and beta-III-tubulin by immunocytochemistry/ immunofluorescence. MiR-122-5p that may regulate Tbr1 expression was predicted by bioinformatics and identified using a Dual-Luciferase assay. The expressions of miR-122-5p and Tbr1 were determined by real-time PCR and western blot before and after the induction. After infection of miR-122-5p, the expressions of Tbr1, NSE, and tauons were measured. BMSCs showed a short spindle shape with a uniform distribution. After 14 days, the induced cells showed neuronal traits with a pyramidal appearance. TargetScan and miRanda showed that miR-122-5p was well complementary with the target site of the Tbr1 3'-untranslated region. Identified by the Dual-Luciferase assay, we found that miR-122-5p could inhibit Tbr1 expression by binding to its 3'-untranslated region. Furthermore, the expressions of Tbr1 mRNA and protein were decreased by real-time PCR and western blot. Overexpression of miR-122-5p downregulated the expressions of Tbr1, NSE, and tauons. MiR-122-5p may negatively regulate Tbr1 expression to affect the differentiation of bMSCs into neuron-like cells. Copyright (C) 2017 The Author(s). Published by Wolters Kluwer Health, Inc.