The anion transport inhibitor DIDS is known to reduce aqueous humor (AH) secretion but questions remain about anion-dependence of the effect. In some tissues, DIDS is reported to cause Na,K-ATPase inhibition. Here, we report on the ability of DIDS to inhibit Na,K-ATPase activity in nonpigmented ciliary epithelium (NPE) and investigate the underlying mechanism. Porcine NPE cells were cultured to confluence on permeable supports, treated with drugs added to both sides of the membrane, and then used for ⁸⁶Rb uptake measurements or homogenized to measure Na,K-ATPase activity or to detect protein phosphorylation. DIDS inhibited ouabain-sensitive ⁸⁶Rb uptake, activated Src family kinase (SFK) and caused a reduction of Na,K-ATPase activity. PP2, an SFK inhibitor, prevented the DIDS responses. In BCECF-loaded NPE, DIDS was found to reduce cytoplasmic pH (pH_i). PP2-sensitive Na,K-ATPase activity inhibition, ⁸⁶Rb uptake suppression and SFK activation were observed when a similar reduction of pH_i was imposed by low pH medium or an ammonium chloride withdrawal maneuver. PP2 and the ERK inhibitor U0126 prevented robust ERK1/2 activation in cells exposed to DIDS or subjected to pH_i reduction but U0126 did not prevent SFK activation or the Na,K-ATPase activity response. The evidence points to an inhibitory influence of DIDS on NPE Na,K-ATPase activity by a mechanism that hinges upon SFK activation associated with a reduction of cytoplasmic pH.