Rab GTPases are important regulators of intracellular trafficking, having distinct intracellular localisation for controlling endocytic, exocytic, and recycling pathways. The impairments in Rab pathways have been linked with drug resistance in various cancers. However, the role of Rab21, an endocytic Rab GTPase, in the emergence of drug resistance is not known. Therefore, we aimed to investigate the role of Rab21 in MRP-1, drug efflux pump, mediated resistance in prostate cancer cells. Drug sensitive (PC-3/Wt) and epirubicin resistant (PC-3/Res) prostate cancer cell lines were used in this study. Our data revealed that PC-3/Res cells had high expression of MRP-1 mRNA and protein in comparison with PC-3/Wt cells. MRP-1 was found distributed between intracellular and cell surface pools in PC-3/Res cells and was capable of drug efflux as shown by doxorubicin and epirubicin efflux assays. Moreover, qRT-PCR and Western blot analysis showed that PC-3/Res cells also had significantly up-regulated expression of Rab21. To study the effect of Rab21 on MRP-1 mediated multidrug resistance, siRNA mediated knockdown of Rab21 was performed. Results showed that the knocking down of Rab21 decreased the drug efflux ability of PC-3/Res cells, possibly by altering the surface localisation of MRP-1. These results suggest that interruption in MRP-1 trafficking to the plasma membrane by Rab21 is a potential strategy to overcome multi drug resistance in cancer cells.