Amphotericin A and cyclosporine are an important antibiotic and immunosuppressive agents, respectively; however, nephrotoxicity is one the main adverse effects. The purpose of this study was to evaluate the effect of inhibiting the p38 MAPK (p38) signaling pathway in nephrotoxicity caused by amphotericin A and cyclosporine from the assessment of stress oxidative, nitric oxide production and cell death in LLC-PK1 and MDCK cell lines. Amphotericin and cyclosporine proved to be oxidative stress (pro-oxidant status), reduction of NO production and DNA fragmentation, determined by flow cytometry using the Propidium iodide dye. In LLC-PK1 cells, the inhibition of the p38 signaling pathway (PD169316 inhibitor) caused a significant reversion status (pro-oxidant to antioxidant), increase of nitric oxide production and reduction in DNA fragmentation when these cells were stimulated with cyclosporine. In contrast, in MDCK cells the same profile was found when these cells were incubated with amphotericin B. Thus, a significant finding of our study is that pharmacological inhibition of p38 MAPK resulted in a substantial reduction in amphotericin B and cyclosporine nephrotoxicity in vitro evaluated by oxidative stress, NO production and cell death parameters. These results suggest that p38 MAPK signaling can contributes to amphotericin B and cyclosporine-induced renal injury in vivo. We also demonstrate that cells from different regions of the nephron (proximal or distal tubules) present varying sensitivities to the toxic effects of amphotericin B and cyclosporine and showed a differentiated participation of p38 MAPK pathway signaling in this process.