Nephrotoxicity is one of the main effects caused by Amphotericin B, the susceptibility of which can vary among nephron segments. The purpose of this study was to evaluate the effect of Amphotericin B on the signaling pathway of protein kinase C (PKC) of VERO and MDCK. Both were incubated with Amphotericin B (2, 4, 6, 8, 10, 15, 20, and 30 μg/mL) for 1, 18, and 24 h, and the cytotoxicty was measured by Neutral Red. In cell signaling, the cell strains were first preincubated with a PKC inhibitor and then exposed to Amphotericin B. Viability was decreased in both cells at 15, 20, and 30 μg/mL of Amphotericin B. However, at 18 and 24 h, the strains presented a different sensitivity. No difference in sensitivity with the involvement of PKC after 1 h could be observed. However, after 18 h, when the effect of Amphotericin B was increased by applying MDCK, the PKC was inhibited. Therefore, involvement of PKC with Amphotericin B can vary among strains from different regions of the nephron. The present study shows the importance of the screening and protecting of drugs at the molecular level in more than one cell strain.