Proton pump inhibitors (PPIs) have become one of the most widely prescribed classes of drugs. However, the use of PPIs is associated with increased risk of acute and chronic kidney disease by mechanisms not entirely known. In the present study, it was found that administration of omeprazole causes a rapid activation of TGF-β/Smad signaling cascade as demonstrated by an increase in TGF-β level and Smad-2/3 phosphorylation in rat kidneys. Activation of TGF-β/Smad signaling cascade is accompanied by an increase in Smad-dependent expression of connective tissue growth factor (CTGF) and tissue inhibitors of matrix metallproteinases-1 (TIMP-1). Interestingly, concomitant administration of the TGF-β inhibitor, disitertide (P144) along with omeprazole markedly inhibits TGF-β/Smad signaling pathway and subsequent CTGF and TIMP-1 expression induced by omeprazole in rat kidneys. Furthermore, omeprazole administration causes a rapid activation of ERK1/2 signaling pathway and subsequent expression of the cell proliferation gene cyclin B1 in rat kidneys. In addition, omeprazole administration significantly induced A disintegrin and metalloproteinase-17 (ADAM17) activity and epidermal growth factor receptor (EGFR) phosphorylation. Interestingly, concomitant administration of the pharmacological inhibitor of EGFR, gefitinib along with omeprazole caused an almost complete reduction of EGFR phosphorylation and subsequent ERK1/2 phosphorylation induced by omeprazole. Moreover, omeprazole administration significantly increased lipid peroxidation and reduced superoxide dismutase (SOD) activity in rat kidneys. Collectively, Omeprazole activates the expression of the profibrotic genes CTGF and TIMP-1 as well as the cell proliferation gene cyclin B1 via reactive oxygen species (ROS)-mediated TGF-β/Smad and ERK1/2 signaling pathways that may contribute to omeprazole-mediated chronic kidney disease.