Polyhistidine is widely used for the delivery of nucleic acids and antibodies into the cell cytoplasm. However, little attention has been concerned on the effect of polyhistidine on the immune system. In this work, we identify a novel function of polyhistidine as an activator of the immune system. Single-molecule fluorescence imaging and single-molecule force measurements show that polybistidine binds specifically to the toll-like receptor 4 (TLR4), inducing receptor dimerization and activation. Moreover, in a B16 melanoma model we demonstrate that polyhistidine treatment inhibits tumor growth in TLR4(+/+) but not TLR4(-/-) mice. These results suggest the potential use of polyhistidine for cancer immunotherapy. (C) 2014 Elsevier Inc. All rights reserved.