The hypersialylated erythropoiesis stimulating agent (ESA) darbepoetin alfa was developed for the treatment of anemia, and has also been reported to have other nonerythropoietic effects. This study outlines one such effect against the toxicity of the radiocontrast medium (RCM) sodium diatrizoate (NaD) in human renal proximal tubular (HK-2) cells in vitro. Using a standard cell viability assay, we observed that pre-incubation of HK-2 cells with darbepoetin (at concentrations of 0.25and 1.0 μg/mL) for 2.5 h prior to addition of NaD (75 mg I/mL, for 2 h) reduced the decrease in cell viability due to the RCM, assayed 22 h after removal of the NaD, whilst maintaining the cells incubated with darbepoetin. Western blot analysis showed that darbepoetin reduced the phosphorylation of c-Jun N-terminal kinases (JNK)1/2 over a period of 1 h incubation with NaD, but did not have an obvious effect on several other targets associated with cell death/survival. However, incubation of HK-2 cells with darbepoetin for a further 22 h after prior exposure to NaD (75 mg I/mL, for 2 h) and subsequent immunoblotting showed that darbepoetin: caused recovery of the activity (phosphorylation) of pro-proliferative/survival signalling molecules, such as Akt (Ser473), STAT (signal transducer and activator of transcription)3(Tyr705); decreased activation of the pro-apoptotic transcription factor FOXO3a by increasing its phosphorylation at Thr32; decreased phosphorylation (activation) of p38 Mitogen activated protein kinase; and reduced poly(ADP-ribose)polymerase (PARP)-1 cleavage. In summary, we present here a beneficial nonerythropoietic effect of darbepoetin alfa against radiocontrast-induced toxicity together with modulation of signalling molecules that play a crucial role in determining cell fate.