Although immunotherapy targeting programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) pathway is being applied in clinic, the response outcomes are heterogeneous, suggesting existences of distinctive subsets within PD-1-expressing T cells that react differently to PD-1/PD-L1 blockade. However, markers to demarcate these subsets in human cancers remain unclear. Here, we found that both PD-1 and B and T lymphocyte attenuator (BTLA) were significantly upregulated on CD4(+) T cells from tumor compared with those from paired non-tumor liver in hepatocellular carcinoma (HCC) patients. Interestingly, over 85% BTLA(+) CD4(+) T cells were PD-1-expressing cells and represented about 50% PD-1(+) CD4(+) T cells in tumors, and that level of BTLA(+)PD-1(+) tumor CD4(+) T cells were selectively associated with advanced stage HCC. BTLA(+) identified highly dysfunctional PD-1-expressing CD4(+) T cell subset, whereas BTLA(-) defined PD-1(+) CD4(+) T cells undergoing activation in HCC. Importantly, blockade of PD-L1 could restore the ability of IFN gamma/TNF-alpha production in BTLA(+)PD-1(+) tumor CD4(+) T cells but partially suppressed the activation of BTLA(-) PD-1(+) CD4(+) T cells. Moreover, we provided evidence that BTLA signals also participated in suppressing CD4(+) T cell function in HCC. In conclusion, BTLA could identify distinct function of PD-1 expressing CD4(+) T cells in human cancer, which might not only advance our understanding of inhibitory receptor blockade, but also provide new targets for clinical predictors of response to these immunotherapies.