Light-activated gene transduction (LAGT) is an approach to localize gene therapy via preactivation of cells with UV light, which facilitates transduction by recombinant adeno-associated virus vectors. Previous studies demonstrated that UVC induces LAGT secondary to pyrimidine dimer formation, whereas UVA induces LAGT secondary to reactive-oxygen species (ROS) generation. However, the empirical UVB boundary of these UV effects is unknown. Thus, we aimed to define the action spectra for UV-induced LAGT independent of DNA damage and determine an optimal wavelength to maximize safety and efficacy. UV at 288, 311 and 320 nm produced significant dose-dependent LAGT effects, of which the maximum (800-fold) was observed with 4 kJ m⁻² at 311 nm. Consistent with its robust cytotoxicity, 288 nm produced significantly high levels of DNA damage at all doses tested, whereas 311, 320 and 330 nm did not generate pyrimidine dimers and produced low levels of DNA damage detected by comet assay. Although 288 nm failed to induce ROS, the other wavelengths were effective, with the maximum (10-fold) effect observed with 30 kJ m⁻² at 311 nm. An in vivo pilot study assessing 311 nm-induced LAGT of rabbit articular chondrocytes demonstrated a significant 6.6-fold (P<0.05) increase in transduction with insignificant cytotoxicity. In conclusion, 311 nm was found to be the optimal wavelength for LAGT on the basis of its superior efficacy at the peak dose and its broad safety range that is remarkably wider than the other UV wavelengths tested.