The mammalian proteome contains multiple proteins encoded by retrovirus- or retrotransposon-related genes, as a consequence of the productive integration of DNA copies of viral RNA molecules into the genomes of host germ cells during early mammalian evolution. The retroviral origin of these proteins is supported by their high similarity to protein domains found in retroviruses, such as capsid-like and protease domains. Among these are the RTL (retrotransposon-like) proteins, including PEG, PNMA, and other RTL family members. We investigated mouse RTL3 (mmRTL3) by expressing both wild-type and mutant forms in mammalian cell lines to study protein expression, cellular effects, localization, proteolytic activity, and interaction partners. We found that mmRTL3 is expressed in mouse heart tissue; the wild-type protein localizes to the cytosol, while the frameshift mutant localizes to the ER. RTL3 has a frameshift efficiency of approximately 30%, lacks proteolytic activity, and forms extracellular viral-like vesicles. It also interacts with proteins involved in metabolic processes and translation.