Establishment of an efficient gene delivery system for human pancreatic beta cells is important for the development of diabetes-targeted cell therapies. The human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vector is well documented to be an effective gene transfer tool for various types of cells. Thus, we examined the efficiency of lentivirus-mediated gene delivery for human islets. Human islets were isolated using defined protocols for enzymatic dissociation and purification using discontinuous Ficoll gradients with a refrigerated Cobe 2991 machine. Isolated islets were shipped to Japan, cryopreserved for 3 months, and then subjected to transduction. A vesicular stomatitis virus G-protein (VSV-G)-pseudotyped lentiviral vector LtV-NLS/LacZ was produced in 293T cells under the Fugene6 method. 804G extracellular matrices were applied for monolayer formation of islets. Detection of NLS/LacZ expression was performed using X-gal staining. Lentiviral transduction was effective in these monolayer islets.