This study examined the branchial epithelium of stenohaline zebrafish Danio rerio, and in particular Na+–Cl− cotransporter‐like 2 (Slc12a10.2)‐expressing ionocytes (Na+–Cl− cotransporter [Ncc]‐cells), which mediate the active uptake of ions from freshwater environments. The study assessed whether the pituitary hormone prolactin (Prl) stimulates the expression of messenger (m)RNAs encoding a Clc Cl− channel family member (clcn2c) and a Na+–K+‐ATPase α1 subunit (atp1a1a.2) expressed in Ncc‐cells. Branchial clcn2c, but not atp1a1a.2 levels, were sensitive to Prl both in vitro and in vivo. These observations suggest that Prl contributes to maintaining systemic Cl− balance via the regulation of clcn2c.