Rods and cones mediate visual perception over 9 log units of light intensities, with both photoreceptor types contributing to a middle 3-log unit range that comprises most night-time conditions. Rod function in this mesopic range has been difficult to isolate and study in vivo because of the paucity of mutants that abolish cone signaling without causing photoreceptor degeneration. Here we describe a novel Gnat2 knockout mouse line (Gnat2-/-) ideal for dissecting rod and cone function. In this line, loss of Gnat2 expression abolished cone phototransduction, yet there was no loss of cones, disruption of the photoreceptor mosaic, nor change in general retinal morphology up to at least 9 months of age. Retinal microglia and Müller glia, which are highly sensitive to neuronal pathophysiology, were distributed normally with morphologies indistinguishable between Gnat2-/- and wildtype adult mice. ERG recordings demonstrated complete loss of cone-driven a-waves in Gnat2-/- mice; comparison to WT controls revealed that rods of both strains continue to function at light intensities exceeding 104 photoisomerizations rod−1 s−1. We conclude that the Gnat2-/- mouse is a preferred model for functional studies of rod pathways in the retina when degeneration could be an experimental confound.