Isothiocyanate is a transient receptor potential ankyrin 1 (TRPA1) agonist and also an inhibitor of ion transporters such as anion exchanger (AE) and Na+/HCO3- co-transporter (NBC). We examined the expression of TRPA1 and ion transporters in monolayers of the rat gastric epithelial cell line RGM1 and investigated the involvement of these factors in the inhibitory action of isothiocyanate on epithelial wound healing. After obtaining a confluent monolayer, a round artificial wound of constant size was induced in the center of the cell monolayer using a pencil-type mixer with a rotating silicon tip. Immediately after the wound induction, cells at the edge of the wound started to form lamellipodia, migrating towards the center of wound, and the cell-free area decreased with time. Addition of allyl isothiocyanate to standard buffer suppressed the recovery of the wound in a concentration-dependent manner without affecting the viability of the RGM1 cells. Icilin, another TRPA1 agonist, dose-dependently inhibited wound repair. Likewise, 4,4’-diisothio- cyanatostilbene-2,2’-disulfonic acid (DIDS), a stilbene compound containing an isothiocyanate group, also inhibited the recovery of epithelial wounds. In addition, the repair of epithelial wounds was suppressed when the cells were incubated in Na+, Cl- or HCO3- free buffer. The RGM1 cells expressed the mRNAs of AE2a and NBC1 but not TRPA1. These results suggested that isothiocyanate impairs the repair of epithelial wounds in RGM1 cells, probably through the inhibition of ion transporters such as AE2a and NBC1 and not the activation of the TRPA1 channel. It is assumed that the process of epithelial repair is associated with the regulation of cell volume and intracellular pH (pHi) by these ion transporters.