Norepinephrine transporter (NET) transfection leads to significant uptake of iodine-131-labeled metaiodobenzylguanidine (¹³¹I-MIBG) in non-neuroendocrine tumors. However, the use of ¹³¹I-MIBG is limited by its short retention time in target cells. To prolong the retention of ¹³¹I-MIBG in target cells, we infected hepatocarcinoma (HepG2) cells with Lentivirus-encoding human NET and vesicular monoamine transporter 2 (VMAT2) genes to obtain NET-expressing, NET-VMAT2-coexpressing, and negative-control cell lines. We evaluated the uptake and efflux of ¹³¹I-MIBG both in vitro and in vivo in mice bearing transfected tumors. NET-expressing and NET-VMAT2-coexpressing cells respectively showed 2.24 and 2.22 times higher ¹³¹I-MIBG uptake than controls. Two hours after removal of ¹³¹I-MIBG-containing medium, 25.4% efflux was observed in NET-VMAT2-coexpressing cells and 38.6% in NET-expressing cells. In vivo experiments were performed in nude mice bearing transfected tumors; results revealed that NET-VMAT2-coexpressing tumors had longer ¹³¹I-MIBG retention time than NET-expressing tumors. Meanwhile, NET-VMAT2-coexpressing and NET-expressing tumors displayed 0.54% and 0.19%, respectively, of the injected dose per gram of tissue 24 h after ¹³¹I-MIBG administration. Cotransfection of HepG2 cells with NET and VMAT2resulted in increased ¹³¹I-MIBG uptake and retention. However, the degree of increase was insufficient to be therapeutically effective in target cells.