The formation of paralogs through gene duplication is a core evolutionary process. For paralogs that encode components of protein complexes such as the ribosome, a central question is whether they encode functionally distinct proteins or whether they exist to maintain appropriate total expression of equivalent proteins. Here, we systematically tested evolutionary models of paralog function using the ribosomal protein paralogsRps27(eS27) andRps27l(eS27L) as a case study. Evolutionary analysis suggests thatRps27andRps27llikely arose during whole-genome duplication(s) in a common vertebrate ancestor. We show thatRps27andRps27lhave inversely correlated mRNA abundance across mouse cell types, with the highestRps27in lymphocytes and the highestRps27lin mammary alveolar cells and hepatocytes. By endogenously tagging the Rps27 and Rps27l proteins, we demonstrate that Rps27- and Rps27l-ribosomes associate preferentially with different transcripts. Furthermore, murineRps27andRps27lloss-of-function alleles are homozygous lethal at different developmental stages. However, strikingly, expressing Rps27 protein from the endogenousRps27llocus or vice versa completely rescues loss-of-function lethality and yields mice with no detectable deficits. Together, these findings suggest thatRps27andRps27lare evolutionarily retained because their subfunctionalized expression patterns render both genes necessary to achieve the requisite total expression of two equivalent proteins across cell types. Our work represents the most in-depth characterization of a mammalian ribosomal protein paralog to date and highlights the importance of considering both protein function and expression when investigating paralogs.