Overexpression of ABCG2 has been reported in cell lines selected for drug resistance and it is widely believed to be important in the clinical pharmacology of anticancer drugs. We and others have previously identified and validated two microRNAs (miRNA; hsa-miR-519c and hsa-miR-520h) targeting ABCG2. In this study, the shortening of the ABCG2 3' untranslated region (3'UTR) was found to be a common phenomenon in several ABCG2-overexpressing resistant cell lines, which as a result removes the hsa-miR-519c binding site and its repressive effects on mRNA stability and translation blockade, thereby contributing to drug resistance. On the other hand, reduced expression of hsa-miR-520h, previously thought to have allowed ABCG2 overexpression, was found to be caused by the sequestering of the miRNA by the highly expressed ABCG2. In drug-sensitive cells, inhibitors against hsa-miR-519c and hsa-miR-520h could augment the cytotoxic effect of mitoxantrone, suggesting a substantial role for both miRNAs in controlling ABCG2 level and thereby anticancer drug response. However, in drug-resistant cells, altering the levels of the two miRNAs did not have any effect on sensitivity to mitoxantrone. Taken together, these studies suggest that in ABCG2-overexpressing drug-resistant cells, hsa-miR-519c is unable to affect ABCG2 expression because the mRNA lacks its binding site, whereas hsa-miR-520h is sequestered and unable to limit ABCG2 expression. Given the recent observation that a truncated 3'UTR is also observed in ABCG2-overexpressing human embryonic stem cells, our results in drug-resistant cell lines suggest that 3'UTR truncation is a relatively common mechanism of ABCG2 regulation.