Cortisol-induced epithelial tightening of a primary cultured rainbow trout gill epithelium model occurs in association with reduced paracellular permeability and increased abundance of select barrier-forming tight junction (TJ) proteins. Corticosteroid receptor (CR) pharmacological blocker studies have suggested that to produce this tightening effect， cortisol acts on the mineralocorticoid receptor (MR) as well as glucocorticoid receptors (GRs). This study considered how cortisol influences model gill epithelium permeability and TJ properties by transcriptional knockdown of the gene encoding the MR (-KD) using double-stranded RNA. Following -KD， a significant reduction in MR protein abundance was observed in the epithelium. The -KD epithelium demonstrated reduced transepithelial resistance (TER) and an increase in the paracellular flux of [H]polyethylene glycol (MW 400?kDa， PEG-400). Concurrently， mRNA abundance of and increased， indicating a possible compensatory response to -KD. Transcript abundance of claudin ()， ， - and decreased while that of increased in -KD preparations. Cortisol-induced epithelial tightening was enhanced in -KD preparations， suggesting that alterations in CRs and TJ composition augmented model epithelium barrier function in response to lowered MR abundance. Cortisol treatment significantly increased the transcript and protein abundance of TJ proteins such as Cldn-8d and -28b. However， in -KD preparations， Cldn-28b protein abundance did not significantly alter in response to cortisol treatment， while Cldn-8d abundance was significantly elevated. Data suggest that -KD compromises normal barrier function of a primary cultured rainbow trout gill epithelium in both the presence and absence of cortisol and that Cldn-28b protein abundance may be modulated by cortisol via the MR only.