Rod photoreceptor neurons in the retina detect scotopic light through the visual pigment rhodopsin (Rho) in their outer segment (OS). Efficient Rho trafficking to the OS through the inner rod compartments is critical for long-term rod health. However, given the importance of protein trafficking to the OS, little is known about the trafficking of rod synaptic proteins. Furthermore, the subcellular impact of Rho mislocalization on rod synapses (i.e. 'spherules') has not been investigated. In this study, we used super-resolution and electron microscopies, along with proteomics, to perform a subcellular analysis of Rho synaptic mislocalization in P23H-Rho-RFP mutant mice. We discovered that mutant P23H-Rho-RFP protein mislocalized in distinct accumulations within the spherule cytoplasm, which we confirmed with adeno-associated virus overexpression. Additionally, we found specific synaptic protein abundance differences in P23H-Rho-RFP mice. Interestingly, in P23H knock-in mice with no RFP tag, we detected no synaptic protein abundance changes. In rd10 mutant rods, Rho mislocalized along the spherule plasma membrane, and there were synaptic protein abundance differences at postnatal day 20. Our findings demonstrate that some rod photoreceptor synaptic proteins are sensitive to Rho mislocalization.