The calcium-sensing receptor (CaSR) is a G protein-coupled receptor responsible for calcium (Ca2+) homeostasis and translating changes of extracellular Ca2+ into intracellular signalling pathways facilitating differentiation and mineralization of osteoblasts in the bone [1]. Odontoblasts are specialised cells in the dental pulp, that produce and regenerate dentin [2]. Increased expression of CaSR in odontoblasts, which was upregulated by elevated extracellular Ca2+, was reported [3], however, whether expression of CaSR changes during differentiation of dental pulp cells when extracellular Ca2+ concentration is constant and how this differentiation influences electrophysiological properties of differentiated odontoblasts remains unclear. We have used immunocytochemistry (ICC) to assess CaSR expression and electrophysiology to measure transmembrane currents, membrane potential and evaluate the cell capacitance in human telomerase reverse transcriptase dental pulp (hTERT DP) cells that were non-differentiated and differentiated in vitro into odontoblasts. hTERT DP-derived odontoblasts demonstrated prominent changes in the cell morphology upon differentiation, so that every single hTERT DP-derived odontoblast occupied lager area than non-differentiated hTERT DP cell without changes of the cell capacitance. Semiquantitative analysis showed that normalized fluorescence intensity of CaSR immunostaining was doubled in differentiated hTERT DP-derived odontoblasts when compared to non-differentiated hTERT DP cells. In addition, increased CaSR expression was evident in cellular processes and on the edges of differentiated odontoblasts. Voltage-clamp experiments showed significantly higher outward transmembrane current densities in differentiated hTERT DP-derived odontoblasts vs. non-differentiated hTERT DP cells at 0.5 mM of extracellular Ca2+. Outward transmembrane current density increased dramatically in the presence of 5 mM Ca2+ in differentiated hTERT DP-derived odontoblasts but not in non-differentiated cells. These data match with current-clamp experiments showing hyperpolarisation of differentiated hTERT DP-derived odontoblasts in response to application of 5 mM extracellular Ca2+ vs. non-significant depolarisation of non-differentiated hTERT DP cells. To conclude, the expression levels and cellular localisation of the CaSR changes during in vitro odontoblast differentiation in stable extracellular Ca2+. Differentiation of hTERT DP into odontoblasts significantly changed cell morphology as well as localization of CaSR expression within the cell. Also, hTERT DP-derived odontoblasts displayed increased sensitivity to extracellular Ca2+ which suggests of its pivotal role during dentinogenesis.