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In vitro transdifferentiation of patient-derived mesenchymal stem/stromal cells (MSCs) into neurons is of special interest for treatment of neurodegenerative diseases. Although there are encouraging studies, little is known about physiological modulations during this transdifferentiation process. Here, we focus on the analysis of gap junction dependent cell-cell communication and the expression pattern of gap junction-building connexins during small molecule-induced neuronal transdifferentiation of human bone marrow-derived MSCs. During this process, the MSC markers CD73, CD90, CD105, and CD166 were downregulated while the neuronal marker Tuj1 was upregulated. Moreover, the differentiation protocol used in the present study changed the cellular morphology and physiology. The MSCs evolved from a fibroblastoid morphology towards a neuronal shape with round cell bodies and neurite-like processes. Moreover, depolarization evoked action potentials in the transdifferentiated cells. MSCs expressed mRNAs encoding Cx43 and Cx45 as well as trace levels of Cx26, Cx37- and Cx40 and allowed transfer of microinjected Lucifer yellow. The differentiation protocol increased levels of Cx26 (mRNA and protein) and decreased Cx43 (mRNA and protein) while reducing the dye transfer. Cx36 mRNA was nearly undetectable in all cells regardless of treatment. Treatment of the cells with the gap junction coupling inhibitor carbenoxolone (CBX) only modestly altered connexin mRNA levels and had little effect on neuronal differentiation. Our study indicates that the small molecule-based differentiation protocol generates immature neuron-like cells from MSCs. This might be potentially interesting for elucidating physiological modifications and mechanisms in MSCs during the initial steps of differentiation towards a neuronal lineage.