Gene transfer offers a substantial promise for the therapy of degenerative ocular diseases. Lentiviral vectors have the ability to efficiently transduce murine photoreceptors during the first days of life, but they are poorly effective on photoreceptors during adulthood. Here, we studied whether a physical barrier was responsible for this impairment. Previous studies have described the capacity of enzymes, such as chondroitinase ABC and neuraminidase X, to modify the structure of the interphotoreceptor matrix (IPM) when subretinally injected. Considering the IPM as a physical barrier that may decrease photoreceptor transduction, we injected different enzymes into the subretinal space of the adult mouse simultaneously with the lentiviral vector preparation, to increase viral transduction by fragilizing the IPM. Subretinal injection of neuraminidase X and chondroitinase ABC induces modifications in the IPM by, respectively, revealing or decreasing peanut agglutinin sites on photoreceptors. The simultaneous subretinal injection of neuraminidase X with a lentiviral vector driving the expression of a reporter gene in the photoreceptors increases the number of transduced cells significantly (around five-fold). After the enzyme treatment, the diffusion of the vector between the pigmented epithelium and the photoreceptors appears to facilitate the lentiviral vector transduction. Such approach targeting the IPM may help to design new strategies to improve gene delivery into the adult photoreceptors.