Soluble programmed death-1 (sPD-1) has been proven to bind PD-1 ligands (PD-Ls), block PD-L/PD-1 interactions, and enhance the cytolysis of cytotoxic T-lymphocytes (CTLs). CH50, a two-domain molecule of fibronectin, has been shown to suppress the development of tumors via enhancing the activity of macrophages. This study evaluated the influence of the recombinant peptide sPD-1-CH50 on antitumor immunity.A secretory recombinant peptide, sPD-1-CH50, containing three functional domains was constructed, and its antitumor effect and mechanism were studied.The data demonstrated that sPD-1-CH50 was able to increase the cytolytic activity of both macrophages and CTLs, especially towards B7-H1-positive tumor cells. The effect correlated with the enhanced production of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma). In vivo studies showed that sPD-1-CH50 inhibited the invasiveness and growth of hepatoma.Regulation of immune cells by sPD-1-CH50 has potential therapeutic value for the inhibition of invasion and growth of hepatoma.