Actinomycin D plays a key role in the successful treatment of Wilms tumour. However, associated liver toxicities remain a drawback to potentially curative treatment. We have used MDCKII cells over-expressing ABCB1, ABCC1, ABCC2 and ABCG2, alongside knockout mouse models to characterise actinomycin D transport and its impact on pharmacokinetics. Growth inhibition, intracellular accumulation and cellular efflux assays were utilised. A 59-fold difference in GI 50 was observed between MDCKII-WT and MDCKII-ABCB1 cells (12.7 nM vs. 745 nM, p < 0.0001). Reduced sensitivity was also seen in MDCKII-ABCC1 and ABCC2 cells (GI 50 25.7 and 40.4 nM respectively, p < 0.0001). Lower intracellular accumulation of actinomycin D was observed in MDCKII-ABCB1 cells as compared to MDCKII-WT (0.98 nM vs. 0.1 nM, p < 0.0001), which was reversed upon ABCB1 inhibition. Lower accumulation was also seen in MDCKII-ABCC1 and ABCC2 cells. Actinomycin D efflux over 2 h was most pronounced in MDCKII-ABCB1 cells, with 5.5-fold lower intracellular levels compared to WT. In vivo studies showed that actinomycin D plasma concentrations were significantly higher in Abcb1a/1b −/− as compared to WT mice following administration of 0.5 mg/kg actinomycin D (AUC 0–6 h 242 vs. 152 μg/L h respectively). While comparable actinomycin D concentrations were observed in the kidneys and livers of Abcb1a/1b −/− and Abcc2 −/− mice, concentrations in the brain were significantly higher at 6 h following drug administration in Abcb1a/1b −/− mice compared to WT. Results confirm actinomycin D as a substrate for ABCB1, ABCC1 and ABCC2, and indicate that Abcb1a/1b and Abcc2 can influence the in vivo disposition of actinomycin D. These data have implications for ongoing clinical pharmacology trials involving children treated with actinomycin D.