Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the adoptive immunotherapy of which is worth studying. CD133, a kind of cancer stem cell (CSC) antigen, together with glypican-3 (GPC3) have been proved to be highly expressed in HCC cells and both of them are used as targets to generate chimeric antigen receptor (CAR) T cells. But there are limitations like “on-target, off-tumor” toxicity, low transfection efficacy and weak antitumor ability in CAR T cells treatment.Methods: First we fused anti-CD133 and anti-GPC3 single chain Fragment variable (scFv) structures with intracellular domains, respectively. Using non-viral minicircle DNA (mcDNA) vectors to generate co-specific CAR T cells (CoG133-CAR T cells) against CD133 and GPC3 double-positive HCC cells. We exhibited the transduction efficiency of CoG133-CAR T cells and the antigen expression of tumor cell lines. Finally, the antitumor efficacy of CoG133-CAR T cells both in vitro and in vivo was detected. Results: GPC3-CAR and CD133-CAR were successfully prepared using non-viral mcDNA vectors to generate effector cells. For the GPC3 and CD133 double-positive HCC (Huh7) xenograft mice, co-specific CAR T cells possessed stronger tumor growth suppression compared to single-targeted CAR (GPC3-CAR and CD133-CAR) T cells which induced only one antigen-mediated signal pathway. The same results also occurred on the in vitro experiments including cytokine secretion, cytotoxicity and proliferation ability of CAR T cells. Vital organs from CoG133-CAR T cells and normal T cells respectively treated Huh7 xenograft mice were stained by hematoxylin and eosin (H&E), the images showed no difference. Conclusions: The mcDNA vectors loading CAR structures were transfected into T cells by electroporation without genetic mutation or mismatch. Huh7 is an HCC cell line with two antigens of GPC3 and CD133 highly expressed. The antitumor efficacy of co-specific CAR (CoG133-CAR) T cells against Huh7 cells is significantly enhanced. The joint design of two specific targets and non-viral vectors leads much more safety, also.