Lentiviral vectors (LVs), which preferentially target nondividing cells, such as neurons, are promising tools for gene therapy. However, these vectors are still unsuitable as they result in insertional mutagenesis. It is therefore essential to prevent insertional mutagenesis if these vectors are to be adopted for safe next generation clinical applications. In order to establish safe genetic therapy with LVs, we focused on the integrase recognition sequence (att) in the long terminal repeat (LTR), which is localized at the edge of the preintegrated viral DNA. We generated LTR-modified LVs (LMLVs), by altering the conserved sequences located just before the cleavage site; this alteration prevented the integration of viral DNA into the host genome. In this study, the LMLVs significantly decreased the LV-mediated transgene expression in HeLa cells compared to the control, i.e., wild-type LTR LVs; this supposedly occurred because integration was prevented. In addition, LMLVs exhibited gene expression in vivo when they were injected into the mouse cerebellum. Moreover, quantitative Alu element-mediated polymerase chain reaction (Alu-PCR), which detects integrated viral DNA, revealed that rate of LMLV-suppressed integration was approximately 1/500-fold compared to that in the case of the wild-type LTR LV. These data suggest that LMLVs efficiently prevent integration as well as exhibit LV-mediated gene expression in mouse cerebellar Purkinje cells in vivo.