The B7-H1/PD-1 pathway has been demonstrated to be involved in tumor evasion. In a previous study, we constructed a eukaryotic expression plasmid (pPD-1A), which expresses soluble PD-1 (sPD-1). In this study, the question of whether or not the blockade of B7-H1 with sPD-1 in vivo and vitro can improve antitumor immunity was investigated. The proliferation of lymphocytes activated by dendritic cells (DCs), which were treated with sPD-1 in vitro, was detected with MTT colorimetry. Mice inoculated with H22 cells were treated by intramuscular injection with pPD-1A. The mRNA expression was analyzed with RT-PCR. The early activation of lymphocytes in vitro was partly improved by sPD-1 blockade. The growth of H22 cells was inhibited significantly after pPD-1A administration. The mRNA expression of 4-1BB, B7.1, IFN-γ and TNF-α of lymphocytes was up-regulated and that of OX40 and IL-10 was down-regulated after pPD-1A administration. Blockade of the PD-1/ B7-H1 pathway with sPD-1 may be a promising strategy for immunotherapy for hepato-carcinoma. Both cytokines and co-stimulatory molecules of lymphocytes could be regulated by sPD-1 blockade.