Multidrug resistance-related protein 2 (Mrp2) is an ATP-driven efflux pump at the luminal membrane in renal proximal tubules. It acts as detoxification mechanism by transporting xenobiotics and metabolic products into urine. The trace element zinc is essential for cellular growth, differentiation and survival. It modulates immune response and is used as dietary supplement. Here, we found that 0.1–10 μM ZnCl2 rapidly stimulated transport of the Mrp2 probe substrate Texas Red (TR) in isolated killifish renal proximal tubules, which provide an established model system to measure efflux transporter activity by using fluorescent probe substrates, confocal microscopy and image analysis. This stimulation was insensitive to the translation inhibitor cycloheximide (CHX), but it was quickly reversed by removing ZnCl2 from the incubation medium. ZnCl2-induced transport stimulation was abolished by inhibitors and antagonists of the endothelin receptor type B (ETB)/nitric oxide synthase (NOS)/protein kinase C (PKC) pathway. Moreover, ZnCl2-induced effects were blocked by inhibition of PKCα using Gö6976 and PKCα inhibitor peptide C2-4. Both the phosphatidylinositol 3-kinase (PI3K) inhibitor LY 294002 and the mammalian target of rapamycin (mTOR) inhibitor rapamycin abolished ZnCl2-induced transport stimulation. Furthermore, the stimulating effects of ZnCl2 were blocked by GSK650394, an inhibitor of the downstream target serum- and glucocorticoid-inducible kinase 1 (SGK1). ZnCl2 also stimulated transport mediated by P-glycoprotein (P-gp) and Breast cancer resistance protein (Bcrp). This is the first report about zinc affecting efflux transporter activity and demonstrates that ZnCl2 triggers a suite of signaling events to evoke a rapid stimulation of ABC transporter-mediated efflux in killifish proximal tubules.