Chimeric antigen receptor (CAR) T cells target specific tumor antigens and lyse tumor cells in an MHC-independent manner. However, the efficacy of CAR-T cell and other cancer immunotherapies is limited by the expression of immune-checkpoint molecules such as programmed death-ligand 1 (PD-L1) on tumor cells, which binds to PD-1 receptors on T cells leading to T cell inactivation and immune escape. Here, we incorporated a PD-L1-targeted single-chain variable fragment (scFv) fusion protein sequence into a CAR vector to generate human anti-PD-L1-CAR-T cells (aPDL1-CART cells) targeting the PD-L1 antigen. Unlike control T cells, aPDL1-CART cells significantly halted the expansion and reduced the viability of co-cultured leukemia cells (Raji, CD46, and K562) overexpressing PD-L1, and this effect was paralleled by increased secretion of IL-2 and IFN-γ. The antitumor efficacy of aPDL1-CART cells was also evaluated in vivo by co-injecting control T cells or aPDL1-CART cells along with PDL1-CA46 cells to generate subcutaneous xenografts in NCG mice. Whereas large tumors developed in mice inoculated with PDL1-CA46 cells alone or together with control T cells, no tumor formation was detected in xenografts containing aPDL1-CART cells. Our data suggest that immune checkpoint-targeted CAR-T cells may be useful for controlling and eradicating immune-refractory hematological malignancies.