AbstractZonula occludens‐1 (ZO‐1), a tight junction protein, contributes to the maintenance of the polarity of odontoblasts and junctional complex formation in odontoblast layer during tooth development. However, expression and possible role of ZO‐1 in human dental pulp cells (hDPCs) during repair process remains unknown. Here, we investigated the expression of ZO‐1 in hDPCs and the relationship with odontoblast differentiation. We found the processes of two adjacent cells were fused and formed junction‐like structure using scanning electron microscopy. Fluorescence immunoassay and Western blot confirmed ZO‐1 expression in hDPCs. Especially, ZO‐1 was high expressed at the cell–cell junction sites. More interestingly, ZO‐1 accumulated at the leading edge of lamellipodia in migrating cells when a scratch assay was performed. Furthermore, ZO‐1 gradual increased during odontoblast differentiation and ZO‐1 silencing greatly inhibited the differentiation. ZO‐1 binds directly to actin filaments and RhoA/ROCK signaling mainly regulates cell cytoskeleton, thus RhoA/ROCK might play a role in regulating ZO‐1. Lysophosphatidic acid (LPA) and Y‐27632 were used to activate and inhibit RhoA/ROCK signaling, respectively, with or without mineralizing medium. In normal cultured hDPCs, RhoA activation increased ZO‐1 expression and especially in intercellular contacts, whereas ROCK inhibition attenuated the effects induced by LPA. However, expression of ZO‐1 was upregulated by Y‐27632 but not significantly affected by LPA after odontoblast differentiation. Hence, ZO‐1 highly expresses in cell–cell junctions and is related to odontoblast differentiation, which may contribute to dental pulp repair or even the formation of an odontoblast layer. RhoA/ROCK signaling is involved in the regulation of ZO‐1.