Classical non-viral methods of gene transfer, such as chemical transfection, have met with limited success of instillation of genetic material into non-proliferating cells in vitro. Among the different kinds of viral vectors, Lentiviral vectors (LVs) have emerged as robust and versatile tool for ex vivo and in vivo gene delivery into multiple cell types including non-dividing cells such as neurons. The capacity of LVs to maintain stable, long-term transgene expression and the substantial flexibility in the design of the expression cassettes account for their increasing use in various pre-clinical and clinical applications. Additionally, LVs have been hugely successful in reprogramming induced pluripotent stem cells (iPSCs). Recent development using LVs in conjunction with a Cre-Lox based reversible system has opened up many new possibilities towards therapeutic application of iPSC technology in various clinical settings. Moreover, improvements in term of biosafety and efficacy, achieved either by modifying the vector design or by involving integration-deficient LVs (IDLVs), have important implications for adoption of LV as the vector of choice for clinical trials. Several human gene therapy clinical trials evaluating the use of LVs for treatment: of human diseases such as Parkinson's disease, β-thalassemia, X-linked adrenoleukodystrophy (ALD), and AIDS are currently ongoing. This review will describe the state of the art achieved by LV technology, its impact on biomedical research, and implications to human clinical trials as therapeutic gene delivery vehicle for a wide range of infectious and genetic diseases.