Several arenaviruses cause hemorrhagic fever disease in humans for which no licensed vaccines are available and current therapeutic intervention is limited to the off-label use of the wide-spectrum antiviral ribavirin. However, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) has proven to be a Rosetta stone for the investigation of virus-host interactions. Arenaviruses have a bisegmented negative-strand RNA genome. The S segment encodes for the virus nucleoprotein and glycoprotein, whereas the L segment encodes for the virus polymerase (L) and Z protein. The ability to generate recombinant LCMV (rLCMV) expressing additional foreign genes of interest would open novel avenues for the study of virus-host interactions and the development of novel vaccine strategies and high-throughput screens to identify antiarenaviral molecules. To this end, we have developed a trisegmented (1L + 2S) rLCMV-based approach (r3LCMV). Each of the two S segments in r3LCMV was altered to replace one of the viral genes by a gene of interest. All r3LCMVs examined expressing different reported genes were stable both genetically and phenotypically and exhibited wild-type growth properties in cultured cells. Reporter gene expression in r3LCMV-infected cells provided an accurate surrogate of levels of virus multiplication. Notably, some r3LCMVs displayed highly attenuated virulence in mice but induced protective immunity against a subsequent lethal challenge with wild-type LCMV, supporting the potential development of r3LCMV-based vaccines.