Indian mustard (Brassica juncea L.) is a plant species used to investigate accumulation of heavy metals. In this study, a hydroponics experiment was conducted to investigate the accumulation of Cu by Indian mustard with application of EDDS, a biodegradable chelator for phytoremediation. The activity of P-type ATPase, a heavy metal ATPase (HMA) responsible for the Cu export, and its transcriptional expression in roots was analyzed. The results showed that increased accumulation of Cu with elevated Cu concentration in medium was only occurred in roots, but not in stems or leaves. The accumulation of Cu in roots, stems and leaves was decreased with elevated EDDS concentration. The results indicated that the uptake of Cu was controlled by plasma membrane of roots and Cu ions rather than Cu-EDDS chelator complexes were taken up by roots. The activity of P-type ATPase in roots was increased with elevated Cu concentration, but decreased with elevated EDDS concentration. The expression pattern of P-type ATPase gene (BjHMA) was in according to the change of its activity. Our results indicated that P-type ATPase might regulate Cu uptake in Indian mustard by actively exporting Cu outside root cells amongst a certain range of ambient Cu concentration.