AbstractCD90 expression and immunoreactive cell localisation in rat dental pulp cells after cavity preparation was investigated. Cavity preparation was performed on the maxillary first molar of 8‐week‐old Wistar rats (n = 36), and immunohistochemistry and quantitative real‐time PCR were performed. CD90‐immunoreactivity was observed among subodontoblastic cells in the control group. One day after cavity preparation, the CD90‐immunoreactivity disappeared under the cavity area. While CD90‐immunoreactivity was faint after 3 days, the re‐arrangement of odontoblasts was detected in contact with dentine. After 5 days, the odontoblasts were observed beneath the dentine, and CD90‐immunoreactive cells were localised under the odontoblast layer. Immunofluorescence showed co‐localisation of CD90 and nestin was detected after 3 days. After 5 days, CD90‐immunoreactivity increased at the subodontoblastic layer. mRNA expression of CD90 and DSPP decreased after cavity preparation, and gradually recovered (P < 0.01). These results suggest that CD90‐immunoreactive cells in the subodontoblastic layer contribute to regeneration of odontoblast and subodontoblastic layers following cavity preparation.