Xenogeneic skin, especially porcine skin, has already been used to cover large wounds in clinic practice of wound care. Our previous data showed that transgenic expression of human cytoxic T-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) in murine skin graft remarkably prolonged its survival in xenogeneic burn wounds without extensive immunosuppression in recipients, suggesting that transgenic hCTLA4Ig expression in skin graft may be an effective and safe method to prolong its survival in xenogeneic wounds for coverage. Lentiviral transgenesis provides an extremely efficient and cost-effective method to produce transgenic animals. However, tissue-targeted transgenic expression of biologically functional protein by lentiviral transgenesis is rarely reported. In this work, a recombinant lentiviral vector (LV), named FKCW in this article, was constructed by inserting a skin-specific hCTLA4Ig expression cassette consisting of keratin 14 (K14) promoter, hCTLA4Ig coding sequence and an intronic fragment. Its efficacy for transgenesis and skin-specific expression of bio-active hCTLA4Ig protein was tested using mice as models. The LV FKCW was readily to be packaged and concentrated to high titres (1.287-6.254 × 10(9) TU/ml) by conventional lentivirus package system. Using eggs collected from only five mated females having been subjected to conventional super-ovulation treatment, 8 hCTLA4Ig transgenic founder mice were generated with the concentrated FKCW vector, and transgenic founder per injected and transferred egg was 6.3%, which was nearly 9-fold higher than that for DNA micro-injection with a similar transgene construct in our previous work. The lentiviral transgenic hCTLA4Ig exhibited strictly skin-specific expression at a level comparable to or even slightly higher than that of transgenic hCTLA4Ig delivered by micro-injection in a similar cassette. Lentiviral transgenic hCTLA4Ig protein remarkably suppressed human lymphocyte proliferation in vitro to a degree comparable to that of commercially purchased purified hCTLA4Ig protein with defined activity at similar concentrations. Besides, lentiviral hCTLA4Ig transgenic mouse skin grafted into rat burn wounds exhibited remarkably extended survival compared to wild-type skin of the same strain (13.8 ± 3.8 vs. 6.8 ± 3.0 days), indicating that lentiviral transgenic hCTLA4Ig did inhibit immune rejection against xenogeneic skin graft in vivo. These results laid down the foundation to further efficiently generate transgenic pigs skin-specifically expressing bio-active hCTLA4Ig by lentiviral transgenesis, and provided a demonstration that transgenic animals with tissue-targeted expression of biologically functional protein can be efficiently produced using LV.