Glycine and/or d-serine are obligatory coagonists of the N-methyl-d-aspartate receptor (NMDAR). Serine racemase, the d-serine-synthesizing enzyme, is expressed by astrocytes and Müller cells of the retina, but little is known about its role in retinal signalling. In this study, we utilize a serine racemase knockout (SRKO) mouse to explore the contribution of d-serine to inner-retinal function. Retinal tissue levels of d-serine in SRKO mice are reduced by 85%. Whole-cell recordings from SRKO retinal ganglion cells showed markedly reduced coagonist occupancy of NMDARs and consequently a dramatic reduction in the NMDAR component of light-evoked responses. NMDAR currents in SRKOs could be rescued by applying exogenous coagonist, but SRKO ganglion cells still displayed lower NMDA/AMPA receptor ratios than wild-type (WT) controls when the coagonist site was saturated. Despite having abnormalities in synaptic glutamatergic transmission, SRKO mice displayed no obvious signs of visual impairment in behavioural testing. These findings raise interesting questions about the role of d-serine in inner-retinal function and development.