Subretinal stimulation of the retina with neurotransmitters, the normal means of conveying visual information, is a potentially better alternative to electrical stimulation widely used in current retinal prostheses for treating blindness from photoreceptor degenerative diseases. Yet, no subretinal electrical or chemical stimulation study has stimulated the OFF and ON pathways differentially through inner retinal activation. Here, we demonstrate the feasibility of differentially stimulating retinal ganglion cells (RGCs) through the inner nuclear layer of the retina with glutamate, a primary neurotransmitter chemical, in a biomimetic way. We show that controlled pulsatile delivery of glutamate into the subsurface of explanted wild-type rat retinas elicits highly localized simultaneous inhibitory and excitatory spike rate responses in OFF and ON RGCs. We also present the spatiotemporal characteristics of RGC responses to subretinally injected glutamate and the therapeutic stimulation parameters. Our findings could pave the way for future development of a neurotransmitter-based subretinal prosthesis offering more naturalistic vision and better visual acuity than electrical prostheses.