The main aim of this study was to evaluate whether microRNA (miRNA) profiling could be a useful tool for in vitro developmental neurotoxicity (DNT) testing. Therefore, to identify the possible DNT biomarkers among miRNAs, we have studied the changes in miRNA expressions in a mixed neuronal/glial culture derived from carcinoma pluripotent stem cells (NT2 cell line) after exposure to methyl mercury chloride (MeHgCl) during the process of neuronal differentiation (2-36 days in vitro (DIV1)). The neuronal differentiation triggered by exposure to retinoic acid (RA) was characterized in the control culture by mRNA expression analysis of neuronal specific markers such as MAP2, NF-200, Tubulin βIII, MAPT-tau, synaptophysin as well as excitatory (NMDA, AMPA) and inhibitory (GABA) receptors. The results obtained from the miRNA expression analysis have identified the presence of a miRNA signature which is specific for neural differentiation in the control culture and another for the response to MeHgCl-induced toxicity. In differentiated neuronal control cultures, we observed the downregulation of the stemness phenotype-linked miR-302 cluster and the overexpression of several miRNAs specific for neuronal differentiation (e.g. let-7, miR-125b and miR-132). In the cultures exposed to MeHgCl (400 nM), we observed an overexpression of a signature composed of five miRNAs (miR-302b, miR-367, miR-372, miR-196b and miR-141) that are known to be involved in the regulation of developmental processes and cellular stress response mechanisms. Using gene ontology term and pathway enrichment analysis of the validated targets of the miRNAs deregulated by the toxic treatment, the possible effect of MeHgCl exposure on signalling pathways involved in axon guidance and learning and memory processes was revealed. The obtained data suggest that miRNA profiling could provide simplified functional evaluation of the toxicity pathways involved in developmental neurotoxicity in comparison with the transcriptomics studies.