Indoxyl sulfate (IS) plays a substantial role in chronic kidney disease (CKD) progression, but there is insufficient understanding of the complex cellular pathways in the kidney. This study investigated the cellular pathways affected by IS in human proximal tubular cells (HK-2 cells). Cell viability, reactive oxygen species (ROS) production and protein expression were analyzed in HK-2 cells exposed to IS. In addition, the effects of catalase and Irbesartan were evaluated. Cell viability of HK-2 cells was decreased following exposure to IS (P=0.001). IS did not significantly increase ROS production compared to cells without IS (P=0.664). Among cellular proteins including ERK, PI3K, Nrf2 and JNK, the expression levels of PI3K and JNK were decreased in IS-exposed cells (P=0.045 and 0.001). In contrast, the expression of HO-1 and NOS3 was increased in proportion to the concentration of IS (P=0.034 and 0.046). In HK-2 cells exposed to 100 μM IS, treatment with catalase and irbesartan increased cell viability, but it was not significant (P=0.190). In addition, use of the drugs did not reverse IS-mediated changes in protein expression for ERK, PI3K, Nrf2 and JNK. In conclusion, cell viability was decreased in HK-2 cells according to IS exposure. This toxicity could involve the activation of ERK, PI3K-AKT, Nrf2-keap1 and JNK pathways as well as NOS3. However, this study could not show the ROS production and the favorable effects of antioxidants and angiotensin-receptor blockers in IS-induced cell injury.