Islet transplantation is a promising therapy for type 1 diabetes, but graft function and survival are compromised by recurrent islet autoimmunity. Immunoprotection of islets will be required to improve clinical outcome. We engineered human β cells to express herpesvirus-encoded immune-evasion proteins, "immunevasins." The capacity of immunevasins to protect β cells from autoreactive T-cell killing was evaluated in vitro and in vivo in humanized mice. Lentiviral vectors were used for efficient genetic modification of primary human β cells without impairing their function. Using a novel β-cell-specific reporter gene assay, we show that autoreactive cytotoxic CD8(+) T-cell clones isolated from patients with recent onset diabetes selectively destroyed human β cells, and that coexpression of the human cytomegalovirus-encoded US2 protein and serine proteinase inhibitor 9 offers highly efficient protection in vitro. Moreover, coimplantation of these genetically modified pseudoislets with β-cell-specific cytotoxic T cells into immunodeficient mice achieves preserved human insulin production and C-peptide secretion. Collectively, our data provide proof of concept that human β cells can be efficiently genetically modified to provide protection from killing mediated by autoreactive T cells and retain their function in vitro and in vivo.