Several approaches have been developed for gene therapy in RPE65-related Leber congenital amaurosis. To date, strategies that have reached the clinical stages rely on adeno-associated viral vectors and two of them documented limited long-term effect. We have developed a lentiviral-based strategy of RPE65 gene transfer that efficiently restored protein expression and cone function in RPE65-deficient mice. In this study, we evaluated the ocular and systemic tolerances of this lentiviral-based therapy (LV-RPE65) on healthy nonhuman primates (NHPs), without adjuvant systemic anti-inflammatory prophylaxis. For the first time, we describe the early kinetics of retinal detachment at 2, 4, and 7 days after subretinal injection using multimodal imaging in 5 NHPs. We revealed prolonged reattachment times in LV-RPE65–injected eyes compared to vehicle-injected eyes. Low- (n = 2) and high-dose (n = 2) LV-RPE65–injected eyes presented a reduction of the outer nuclear and photoreceptor outer segment layer thickness in the macula, that was more pronounced than in vehicle-injected eyes (n = 4). All LV-RPE65–injected eyes showed an initial perivascular reaction that resolved spontaneously within 14 days. Despite foveal structural changes, full-field electroretinography indicated that the overall retinal function was preserved over time and immunohistochemistry identified no difference in glial, microglial, or leucocyte ocular activation between low-dose, high-dose, and vehicle-injected eyes. Moreover, LV-RPE65–injected animals did not show signs of vector shedding or extraocular targeting, confirming the safe ocular restriction of the vector. Our results evidence a limited ocular tolerance to LV-RPE65 after subretinal injection without adjuvant anti-inflammatory prophylaxis, with complications linked to this route of administration necessitating to block this transient inflammatory event.